ABSTRACT

Title of Document: INTERVENTION STRATEGIES TO

REDUCE FOODBORNE PATHOGENS IN POULTRY DURING GROW-OUT AND PROCESSING

Rommel Max T.S.L. Tan, M.S., 2008 Directed By: Associate Professor, Nathaniel L. Tablante,

Department of Veterinary Medicine VA-MD Regional College of Veterinary Medicine University of Maryland-College Park

Several foodborne pathogens like Salmonella spp., Campylobacter jejuni and

Clostridium perfringens can occasionally be traced to poultry sources. The

development of intervention strategies that are applicable to different stages of

poultry production can help lessen the level of these pathogens in poultry by-products

and hence, reduce the incidence of poultry-borne food poisoning. In the present study,

the efficacy of Poultry Litter Treatment® in reducing Clostridium perfringens counts

in poultry litter was investigated. The effect of windrow-composting in reducing

microbial load in poultry litter was also studied. In addition, a study of bacterial

profiles in a poultry processing line was conducted. Finally, the efficacies of two

online reprocessing antimicrobials in reducing bacterial pathogen load were

compared.

INTERVENTION STRATEGIES TO REDUCE FOODBORNE PATHOGENS IN POULTRY DURING GROW-OUT AND PROCESSING

By

Rommel Max T.S.L. Tan

Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment

of the requirements for the degree of Master of Science

2008 Advisory Committee: Associate Professor Nathaniel L. Tablante, Chair Associate Professor Y. Martin Lo Dr. Daniel A. Bautista Dr. Bernard D. Murphy

© Copyright by Rommel Max T.S.L. Tan

2008

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Acknowledgements

I am greatly indebted to Dr. Nathaniel Tablante, my thesis adviser. He provided me with the graduate teaching assistantship when I changed my plans during the course of my Masters degree program in the department. He provided help in arranging possible projects with Lasher Laboratory. He taught me to drive which I needed in my thesis work. He is very humble and affable, endeared by all the students in the department as well as in the undergraduate course that he teaches. He always lends an ear even if he is very busy. He is very patient even though I have made many mistakes. He was very generous in his recommendations. He has, in effect, saved my career from total wreck. I am also greatly indebted to Dr. Daniel Bautista, the Director of Lasher Poultry Diagnostic Laboratory and one of my thesis committee members. He, along with Lasher Laboratory, provided me with the funding for projects to help me finish my thesis. In addition, he provided lodging and transportation while I was undertaking some of my research on the Eastern Shore. I am very thankful to Drs. Y. Martin Lo and Bernard Murphy, the two other members of my thesis committee, for their very valuable and helpful inputs to improve my thesis. I would like to thank Dr. Siba Samal, the Department Chair, and the Department of Veterinary Medicinefor providing me with a departmental graduate assistantship. I would like to thank Jones Hamilton, Co. for funding one of my thesis projects. I am very much indebted to Lasher Poultry Diagnostic Laboratory staff and personnel (Dr. Daniel Bautista, Ms. Kathy Phillips, Ms. Brenda Sample, Ms. Billie Jean Wright, Ms. Luanne Sullivan, Ms. Courtney, Mr. Colby Smith and Mr. Stephen Collier) for helping me with a sizable part of my thesis project, without which I would not have been able to do on my own. I want to thank Mr. George W. Malone, who undertook the actual windrowing procedure and sample collection and provided pictures of such. I am also very much indebted to Dr. Ana Baya of the Maryland Department of Agriculture (MDA) Animal Health Laboratory in College Park for allowing me to conduct part of my research in her laboratory. I also want to express my gratitude to the MDA-College Park staff (especially Ms. Laura Smith) who helped me with a portion of my studies.

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I would like to thank Dr. Suman Mukhopadhyay’s laboratory (especially Mr. Arindam Mitra) for allowing me to use some of their equipment. I would like to thank my Biometrics professor, Dr. Frank Siewerdt, who taught and assisted me with statistical methods to analyze my data. I would also like to thank Dr. Susan White of the University of Delaware, who developed the study design for the litter amendment study. Finally, I would like to thank the professors, post-doctoral researchers, and classmates in the department for all their help and support.

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Table of Contents Acknowledgements ...................................................................................................ii Table of Contents .....................................................................................................iv List of Figures ..........................................................................................................vi Chapter 1: Introduction..............................................................................................1

1.1 Common Foodborne Pathogens in Poultry..................................................1 1.2 Hazard Analysis and Critical Control Points...............................................5 1.3 Research Rationale.....................................................................................7 1.4 Research Objectives ...................................................................................8

Chapter 2: Descriptive Study of the Microbial Profile of Poultry Litter from Chronically Affected Gangrenous Dermatitis Farms ................................................10

2.1 Review of Literature ......................................................................................10 2.1.1 Gangrenous Dermatitis ...........................................................................10 2.1.2 Current Litter Survey Studies ..................................................................11

2.2 Materials and Methods...................................................................................12 2.2.1 Sample Size and Collection .....................................................................12 2.2.2 Bacterial Enumeration ............................................................................13 2.2.3 Statistical Analysis ..................................................................................13

2.3 Results and Discussion...................................................................................13 Chapter 3: An Evaluation of Poultry Litter Treatment® (PLT®) on Clostridium spp. Recovery from Poultry Litter ...................................................................................17

3.1 Review of Literature ......................................................................................17 3.1.1 Clostridial Diseases in Poultry ................................................................17 3.1.2 Poultry Litter Amendment .......................................................................18

3.2 Materials and Methods...................................................................................22 3.2.1 Broiler House Layout and Design ...........................................................22 3.2.2 Litter Sampling and Bacterial Enumeration ............................................24 3.2.3 Statistical Analysis ..................................................................................25

3.3 Results and Discussion...................................................................................25 Chapter 4: Effect of Windrow Composting on the Microbiological Profile of Poultry Litter .......................................................................................................................32

4.1 Review of Literature ......................................................................................32 4.2 Materials and Methods...................................................................................34

4.2.1 Windrow Construction and Litter Sampling.............................................34 4.2.2 Bacterial Enumeration ............................................................................35 4.2.3 Statistical Analysis ..................................................................................36

4.3 Results and Discussion...................................................................................36 Chapter 5: A Baseline Study of the Level of Bacterial Foodborne Pathogens at Different Stages of Poultry Processing.....................................................................41

5.1 Review of Literature ......................................................................................41 5.2 Materials and Methods...................................................................................52

5.2.1 Sample Collection ...................................................................................52 5.2.2 Bacterial Enumeration. ...........................................................................53

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5.2.3 Statistical Analysis ..................................................................................54 5.3 Results and Discussion...................................................................................54

Chapter 6: Comparison of Two Online Reprocessing (OLR) Antimicrobials...........68 6.1 Review of Literature ......................................................................................68 6.2 Materials and Methods...................................................................................72

6.2.1 Sample Size and Collection .....................................................................72 6.2.2 Bacterial Enumeration ............................................................................73 6.2.3 Statistical Analysis ..................................................................................73

6.3 Results and Discussion...................................................................................74 Chapter 7: Summary and Conclusion .......................................................................78 Appendices..............................................................................................................81 Bibliography.......................................................................................................... 106

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List of Figures

Figure 1. Average total aerobic, coliform and Clostridium perfringens levels in

litter of gangrenous dermatitis-affected and control broiler farms

Figure 2. Layout of the litter amendment experimental houses

Figure 3. Compartmentalization of the litter amendment experimental houses

into blocks and location (Blocks/Location)

Figure 4. Randomized complete block design of the litter amendment houses

Figure 5. Average Clostridium perfringens counts in the east house

Figure 6. Average Clostridium perfringens counts in the west house

Figure 7. Least square means of Clostridium perfringens counts in soil during

harvest at the west house

Figure 8. Average total aerobic, coliform and Clostridium perfringens

counts in litter pre- and post-composting

Figure 9. Basic layout of a poultry processing plant

Figure 10. Line chart of average coliform counts of carcass rinses taken from

different stages of a poultry processing plant

Figure 11. Post-scald carcass rinse total aerobic, coliform, and E. coli counts

shown in previous studies

Figure 12. Post-pick carcass rinse total aerobic, coliform, and E. coli counts

shown in previous studies

Figure 13. Post-evisceration carcass rinse total aerobic, coliform, and E .coli

counts shown in previous studies

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Figure 14. Post-IOBW carcass rinse total aerobic, coliform, and E .coli counts

shown in previous studies

Figure 15. Post-chill carcass rinse total aerobic, coliform, and E .coli counts

shown in previous studies

Figure 16. Line chart of average Campylobacter spp. counts of carcass rinses

taken from different stages of a poultry processing plant

Figure 17. Average total aerobic counts of carcass rinses taken from pre-OLR,

post-OLR, and post-chill stages of the processing plant using

SANOVATM antimicrobial

Figure 18. Average total aerobic counts of carcass rinses taken from pre-OLR,

post-OLR, and post-chill stages of the processing plant using

Perasafe® antimicrobial

Figure 19. Average coliform levels of carcass rinses taken from pre-OLR, post-

OLR, and post-chill stages of the processing plant using SANOVATM

antimicrobial

Figure 20. Average coliform counts of carcass rinses taken from pre-OLR, post-

OLR, and post-chill stages of the processing plant using Perasafe®

antimicrobial

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Chapter 1: Introduction

1.1 Common Foodborne Pathogens in Poultry

Escherichia coli is part of the normal bacterial flora of warm blooded animals

and humans. Although generally considered as commensal organisms,

enteropathogenic strains of E. coli have been reported. Currently there are six

different diarrheic groups of E. coli namely: Enteropathogenic (EPEC),

Enterotoxigenic (ETEC), Enteroinvasive (EIEC), Enterohemorrhagic (EHEC),

Enteroaggregative (EAEC), and Diffusely adherent (DAEC) (Fratamico et al, 2002).

Avian pathogenic E. coli (APEC), found in intestines of healthy birds are mainly

EPEC and ETEC (Kariuki et al, 2002). In the study by Kariuki et al (2002), 32.5% of

fecal swabs taken from apparently healthy chickens and enriched for E. coli detection

were positive for hybridization with eae gene of EPEC and 13.3% were positive for

hybridization with lt, st1 and st2 gene of ETEC. However, according to Fratamico et

al (2002), the EPEC serotypes found in animals are not usually associated with

human infection. Asymptomatic humans are mainly the reservoirs of these EPEC

while foods that are served cold are the main source of ETEC outbreaks (Fratamico et

al, 2002). Because of the severity of diseases (hemorrhagic colitis and hemolytic-

uremic syndrome) that EHEC serotype O157:H7 cause, special attention has been

given to this particular serotype in food safety studies. EHEC have a characteristic of

being able to produce different types of Shiga toxins (Meng et al, 2001). Serotype

O157:H7 is the predominant cause of EHEC-associated diseases in humans in the

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United States, Canada, the United Kingdom, and Japan (Meng et al, 2001).

Undercooked ground beef (33.1%) remains the main vehicle for O157:H7 outbreaks

(Meng et al, 2001). The prevalence of Shiga toxin producing E. coli in broilers is very

low or absent (Beutin et al, 1993; Kobayashi et al, 2002). In the 1994-1996

Nationwide Broiler Chicken Microbiological Baseline Data Collection Program

results, none of the 1,297 broilers tested was positive for E. coli O157:H7 (FSIS,

1996a). E. coli detection and enumeration is generally used as an indicator of recent

fecal contamination or unsanitary food processing (Feng et al, 1998).

Salmonella spp. are facultative anaerobic, non-lactose fermenting members of

the family Enterobacteriaceae. Because of its characteristic resistance and uncanny

ability to adapt in extreme environmental conditions (low pH, high CO2, high

temperature, high salt concentration), Salmonella spp. poses a great concern in food

safety (D’Aoust et al, 2001). Salmonella serotype is based on capsular, flagellar, and

envelop antigens (Gray and Fedorka-Cray, 2002). According to the 2005 Salmonella

Annual Summary of the Center for Disease Control (CDC, 2007), the five most

commonly reported serotypes from human cases are Typhimurium (19.3%),

Enteritidis (18.6%), Newport (9.1%), Heidelberg (5.3%), and Javiana (3.7%).

Foodborne salmonella cases are most commonly associated with chicken

consumption (Gray and Fedorka-Cray, 2002). This is due to the asymptomatic

intestinal carriage in chickens which would ultimately lead to contamination of

carcasses during slaughter (Gast, 2003a). However, it should be noted that it is not the

host-adapted serotypes (S. pullorum and S. gallinarum) that cause foodborne

outbreaks but the paratyphoid ones (Gray and Fedorka-Cray, 2002). According to the

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serotype profiling study on meat and poultry products by the United States

Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) for

2006 (FSIS, 2007b), the five most commonly reported serotypes from broiler sources

are Kentucky (48.97%), Enteritidis (13.66%), Heidelberg (11.34%), Typhimurium

(8.08%), and serotype 4,5,12:i:- (4.30%). Salmonella spp. possess three virulence

factor toxins: endotoxin which causes fever, heat labile enterotoxin which causes

secretory diarrhea, and heat stable cytotoxin which causes protein synthesis inhibition

and subsequent epithelial cell damage (Gast, 2003b). Salmonella infection can cause

four possible disease patterns namely: gastroenteritis, enteric fever, bacteremia with

or without focal extraintestinal infection, and asymptomatic carrier (Gray and

Fedorka-Cray, 2002).

Campylobacter jejuni subsp. jejuni are gram negative, curved rod bacteria

with polar flagella often found in poultry. Avians are the most common host species

for Campylobacter because of the high body temperature of birds (Keener et al,

2004). Campylobacteriosis is the most common foodborne bacterial illness in the U.S.

accounting for an estimated 2.5 million cases annually (Mead et al, 1999). Although

Campylobacter spp. are known to be susceptible to low pH, the infectious dose

appears to be <1000 bacteria, considering it will be passing through gastric acids

(Nachamkin, 2001). Unlike Salmonella spp., Campylobacter spp. is typed using the

Penner HS (heat stable) serotyping scheme which is determined by a capsular

polysacharride (Nachamkin, 2001). Campylobacter jejuni, C. coli, and C. lari account

for more than 99% of the human isolate with C. jejuni constituting 90% (Hunt et al,

1998). Among the different severe sequelae that are associated with C. jejuni

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infection are Guillain-Barŕe syndrome (neurologic syndrome), Reiter’s syndrome

(sterile reactive arthritis), Miller Fisher syndrome, septic arthritis, osteomyelitis, and

chronic recurrent diarrhea (Altekruse and Swerdlow, 2002; Keener et al, 2004).

Unlike Salmonella, there is no standard subtyping scheme for Campylobacter. Most

sporadic outbreaks peaking in summer months are associated with consumption of

undercooked poultry or other foods that were cross-contaminated by raw poultry

(Alterkruse and Swerdlow, 2002). Among the many virulence factors of

Campylobacter are mucous colonization, flagellar attachment, iron acquisition, host

cell invasion, and toxin production (Alterkruse and Swerdlow, 2002).

Clostridium perfringens are gram positive, spore-forming, anaerobic bacteria.

According to Mead et al (1999), the estimated total cases of foodborne Clostridium

perfringens intoxication are 250,000 annually while causing only about 41

hospitalizations. The likely explanation for this minimal hospitalization is localized

damage to villus tip cells caused by the organism and the consequently normal

epithelial turnover caused by diarrhea (Labbe and Juneja, 2002; McClean, 2001).

Because of the mild and indistinguishing characteristic symptom of Clostridium

perfringens type A food poisoning, most cases are not recognized and reported

(McClane, 2001). Another likely explanation is that the infectious dose of

Clostridium perfringens is very high (about 106 to 107 vegetative cells per gram of

food) because of its susceptibility to gastric acid (McClane, 2001). However,

Clostridium perfringens incidence in grow-out farms and in processed poultry

remains high (Craven, 2001; Craven et al, 2001). Craven et al (2001) isolated Cl.

perfringens in 94% of fecal samples and in 81% of post-chill carcass samples from

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poultry flocks tested. Of particular concern in poultry with respect to foodborne Cl.

perfringens poisoning are large roaster broilers and turkeys because of the difficulty

of attaining high internal temperatures during cooking (McClane, 2001). Because of

the relative heat tolerance of Clostridium perfringens vegetative cells, the heat

resistance of its spores, and its ability to rapidly multiply (doubling in less than 10

minutes), temperature abuse during improper holding of foods is a major contributor

to Clostridium perfringens type A poisoning (McClane, 2001). The toxin responsible

for the characteristic symptom of Cl. perfringens type A poisoning is CPE

(Clostridium perfringens enterotoxin). One unique characteristic of Clostridium

perfringens intoxication is that the enterotoxin is released along with spores during

sporangial autolysis (Labbe and Juneja, 2002). Another major virulence factor of

Clostridium perfringens is the alpha toxin which is present in all toxin types (A-E).

The alpha toxin is actually a phospholipase C which breaks down the lecithin in the

cell membrane producing tissue breakdown (Labbe and Juneja, 2002).

1.2 Hazard Analysis and Critical Control Points

In 1996, the United States Department of Agriculture (USDA) Food Safety and

Inspection Service (FSIS) published the final rule of Pathogen Reduction: the Hazard

Analysis and Critical Control Points (HACCP) System. This new regulation required

all slaughter and processing plants to adopt HACCP as a system of process control,

follow a Standard Operating Procedure (SOP) for sanitation, conduct daily microbial

testing for generic E. coli to verify process control, and set pathogen reduction

performance standards for generic E. coli and Salmonella spp. (FSIS, 1996b).

HACCP is a system where processing establishments identify the food safety hazards,

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institute controls to prevent those hazards, monitor the performance of those controls,

and maintain records for assessment (FSIS, 1996b). HACCP has seven guiding

principles namely: hazard analysis, critical control points (CCP) identification,

establishment of critical limits, monitoring procedures, corrective actions,

recordkeeping, and verification procedures (FSIS, 1996b). Hazard analysis is the

identification of the food safety hazard on each process control and the preventive

measures to control such hazards (FSIS, 1996b). A critical control point is a

procedure or step where control can be applied and a food safety hazard can be

prevented, eliminated, or reduced to an acceptable level (FSIS, 1996b). Critical limit

is the maximum or minimum value which a CCP must meet to prevent a food safety

hazard (FSIS, 1996b). Monitoring involves whether the critical limits are being met,

and corrective actions refer to measures taken if there is a deviation from the critical

limits (FSIS, 1996b). Proper record keeping and periodic verification of the HACCP

system are used to determine whether the system complies with the HACCP plan

(FSIS, 1996b). In addition to mandating the HACCP system, the FSIS also set up

pathogen reduction performance standards for generic E. coli and Salmonella spp.

based on the baseline study done by the Nationwide Broiler Chicken Microbiological

Baseline Data Collection Program from July 1994 through June 1996 on raw post-

chill carcass rinses (FSIS, 1996a; FSIS, 1996b). The 80th and 98th percentile of

generic E. coli levels seen in broilers were 80 and 1100 cfu/ml, respectively (FSIS,

1996b). These results rounded to the closest power of 10 (100 and 1000) were

adopted as the minimum acceptable and marginally acceptable limit, respectively

(FSIS, 1996b). The Salmonella spp. prevalence result was 20% (FSIS, 1996b). A

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more current baseline study done by the Nationwide Young Chicken Microbiological

Baseline Data Collection Program from November 1999 through October 2000

showed lower baseline levels. The 80th and 98th percentile of generic E. coli levels

seen in broilers in this study were 32 and 390 cfu/ml, respectively (FSIS, 2005).

Salmonella spp. prevalence, likewise, was lower at 8.7% (FSIS, 2005). Another

baseline study is currently being done (FSIS, 2007a) which aims to produce baseline

for Salmonella spp. and Campylobacter spp. counts. According to the baseline study

conducted by Berrang et al (2007) in 20 processing plants, the overall mean count for

Campylobacter spp. in raw post-chill carcass rinse is 0.43 log10 cfu/ml.

1.3 Research Rationale

According to Mead et al (1999), the estimated annual total cases of

Salmonella spp. and Campylobacter spp. foodborne illnesses are 1.4 million and 2.5

million, respectively, making up more than half of the estimated 5.2 million bacterial

foodborne illness cases. Salmonella cases cause approximately 16,400

hospitalizations and 600 deaths, while Campylobacter cases cause approximately

13,200 hospitalizations and 100 deaths (Mead et al, 1999). According to the USDA

Economic Research Services (2003), the annual economic cost of Salmonella spp.

and Campylobacter spp. foodborne cases are $2.9 billion and $1.2 billion,

respectively. Based on the epidemiological data reported by the U.S. Centers for

Disease Control in 2006, the preliminary FoodNet data for 10 States showed that

there were 6,655 Salmonella cases and 5,712 Campylobacter cases. The importance

of continuous surveillance was highlighted by Zhao et al (2001) who conducted a

survey of bacterial contamination around the Greater Washington, D.C. area on retail

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raw meat. They found that 70.7% of the chicken carcasses sampled were positive for

Campylobacter spp., 38.7% were positive for E. coli, and 4.2% were positive for

Salmonella spp. Even if the final cooking process assures the killing of these common

foodborne pathogens, the results of these descriptive studies show the possible source

of infection if proper cooking practices are not followed. Evidently, foodborne

illnesses remain the primary concern for the general public, public health officials,

and the food production industry. Employment of effective intervention strategies by

applying the “multiple hurdle approach” (Russell 2007c) to reduce the level of

foodborne bacterial pathogens during food production and processing is therefore

critical in minimizing foodborne illnesses.

1.4 Research Objectives

The main goal of this research was to evaluate the effectiveness of various

intervention strategies for pathogen reduction employed by poultry companies during

different stages of production and processing. This research was focused only on the

grow-out and processing aspects of a vertically integrated poultry production system.

On the production side, the objective of the descriptive study was to ascertain whether

Clostridium perfringens counts between gangrenous dermatitis positive farms were

higher than those of negative farms. Results from this study could serve as a model on

how a poultry disease, namely gangrenous dermatitis, can be a possible source of

foodborne pathogens for humans. The objective of the PLT® study was to evaluate

the efficacy of sodium bisulfate in reducing the load of Clostridium perfringens in

poultry litter, a potential source of foodborne pathogens. The objective of the

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composting-windrow study was to evaluate the ability of the windrow technique to

decrease bacterial pathogen load in poultry litter that is bound to be recycled for the

next grow-out cycle. On the processing side, a simple line study was carried out with

two objectives in mind. First, the levels of bacterial pathogen reduction at different

stages of the poultry processing line were characterized. Secondly, the baseline

pathogen counts of the poultry farm involved in the present study was established to

provide the farm manager with information for improving management schemes. The

comparative online reprocessing antimicrobial study also carried two specific

objectives. The first objective was to compare the efficacy of two antimicrobials in

reducing bacterial pathogens in poultry carcasses. This data is extremely important

for processing managers in deciding whether it is cost-effective to upgrade or change

the antimicrobials currently being used as an online reprocessing chemical. The

second objective was to compare the level of bacterial pathogens in the two

evisceration lines and assess whether standards were being met.

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Chapter 2: Descriptive Study of the Microbial Profile of Poultry Litter from Chronically Affected Gangrenous

Dermatitis Farms

2.1 Review of Literature

2.1.1 Gangrenous Dermatitis

Gangrenous dermatitis is caused by Clostridium septicum, Clostridium

perfringens type A, Clostridium novyi, Clostridium sordelli, and Staphylococcus

aureus infection (Wilder et al, 2001). It occurs in broiler chickens at 17 days to 20

weeks of age but mostly at 4-8 weeks of age (Wages and Opengart, 2003a). Neumann

et al (2006) reported that 23.5% of gangrenous dermatitis lesions sampled were

positive for Cl. perfringens, 41.2% were positive for Cl. Septicum, and 29.4% were

positive for both. Although Clostridium perfringens is commonly implicated as the

cause of gangrenous dermatitis, there have been published case reports of

Staphylococcus-induced (Cervantes et al, 1988) and Clostridium septicum-induced

(Willoughby et al, 1996) cases. In an experimental in vivo model study, Wilder et al

(2001) observed that Staphylococcus aureus and Clostridium septicum had either a

synergistic or an additive effect. In one combination, the mortality was high but was

zero when either organism was inoculated alone (synergistic). In another

combination, the mortality was high, but when inoculated alone, the Staphylococcus

aureus strain caused low mortality (additive). It is thought that gangrenous dermatitis

is a consequence of immunosuppressive viral infections such as Infectious Bursal

Disease (IBD), Chicken Anemia Virus (CAV), reticuloendotheliosis virus, and

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Inclusion Body Hepatitis virus (IBH) (Wages and Opengart, 2003a). With CAV and

avian reovirus infection, gangrenous dermatitis occurs secondary to the hemorrhagic

lesions caused by blue wing disease (Wages and Opengart, 2003a). Other

management related factors leading to wounds or weakened skin associated with

gangrenous dermatitis are caponization, wet litter, feed outages, and overcrowding

(Wages and Opengart, 2003a). In addition to these, Clark et al (2004) enumerated

some factors that may ultimately cause hysteria and subsequent cuts from the

scratches: lightning storms, longer day length, increased lighting intensity, light

restriction program, and low dietary sodium. Gross lesions consist of moist,

gangrenous skin; subcutaneous edema and emphysema; and skeletal muscular

necrosis and hemorrhage (Wages and Opengart, 2003a). Hepatic, splenic, renal, and

pulmonary lesions may be present. Since Clostridium spp. are very durable due to

their spore forming chararcteristic, the main strategy suggested by Clark et al (2004)

is to keep the number of Clostridium spp. as low as possible in order to allow faster

recovery.

2.1.2 Current Litter Survey Studies

The microbiological profiles of poultry litter are an important consideration

because of the constant contact of poultry with litter in non-elevated floor-type houses

that are commonly used in the United States. Previous microbiological surveys and

profiles of poultry litter have been conducted (Martin et al, 1998; Hartel et al, 2000;

Vizzier Thaxton et al, 2003; Terzich et al, 2000; Omeira et al, 2006; Craven et al,

2001). The studies by Terzich et al (2000) and Vizzier Thaxton et al (2003) only

involved total gram positive and total anaerobic counts, respectively, and not a

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specific count of Clostridium perfringens. Omeira et al (2006) did specific

Clostridium perfringens counts but their litter samples were taken from healthy farms.

Craven et al (2001), on the other hand, did an incidence survey of Clostridium

perfringens and found 23% of the 412 litter samples to be positive. The objective of

the present study was to describe the levels of Clostridium perfringens in farms

chronically affected with gangrenous dermatitis as compared to gangrenous

dermatitis free (control) farms as a way to show how gangrenous dermatitis farms

may pose a threat to food safety. This is important because several millions of

Clostridium perfringens are required to produce typical foodborne illnesses, making

species-specific enumeration studies necessary to more accurately describe the food

safety implication of gangrenous dermatitis affected farms (Shahidi and Ferguson,

1971).

2.2 Materials and Methods

2.2.1 Sample Size and Collection

Ten litter samples were taken from each of eight farms chronically affected

with gangrenous dermatitis as well as from three control farms (negative for

gangrenous dermatitis). Samples were collected as follows: three from the side walls,

four from the drinkers, and three from the center of the house. Sampling was

performed by scooping a handful of litter and placing it in a sealable plastic bag. The

samples were then shipped overnight for quantification to the Maryland Department

of Agriculture Animal Health Laboratory in College Park, Maryland.

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2.2.2 Bacterial Enumeration

Twenty-five grams of soil samples were dissolved in 225 ml PBS (Sigma-

Aldrich Corp., St. Louis, MO) to make a 1:10 solution. The resulting solution was

shaken for at least 15 minutes. Bacterial quantification was performed by spread

plating using MacConkey agar (Becton, Dickenson and Company, Sparks, MD) for

coliform count, Trypticase Soy agar (Becton, Dickenson and Company, Sparks, MD)

for total aerobic count, and Shahidi-Ferguson Perfringens (SFP) agar (Becton,

Dickenson and Company, Sparks, MD) for Clostridium pefringens count. Only three-

fold 1:10 dilutions were made. The SFP agar (Shahidi and Ferguson, 1971) culture

plates were sealed in a plastic bag using a plastic sealer with an AnaeroPak®

(Mitsubishi Gas Chemical, Inc., New York, NY) included inside. Bacterial

quantification was performed after 20-24 hours of incubation.

2.2.3 Statistical Analysis

All counts were transformed to log form. Using Statistical Analysis

System/SAS (SAS Institute Inc., Cary, NC), PROC GLM (Generalized Linear Model)

with nested arrangement was employed to analyze whether there was a statistical

difference between Clostridium perfringens counts from chronically affected

gangrenous dermatitis farms and normal (control) farms at α=0.05.

2.3 Results and Discussion A total of eight poultry houses were sampled as gangrenous dermatitis

affected farms and three as gangrenous dermatitis negative (control) farms (Figure 1

and Appendix A). The bacterial load of gangrenous dermatitis affected farms was

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consistently higher than that of control farms for TAC (total aerobic count), coliform

counts, and Clostridium perfringens counts. The differences in all counts between

farms nested under their farm status category (affected or control) were all

statistically significant (Table 2). While the difference in TAC and coliform counts

between affected and control farms were statistically insignificant, the difference in

Clostridium perfringens counts was statistically significant.

The total aerobic counts for both affected and control farms were

approximately 105 to 107 log10 cfu/ml. These counts coincide with the litter survey

studies of Martin et al (1998) and Vizzier Thaxton et al (2003) but were less than that

of the litter survey study of Terzich et al (2000). The difference in TAC between

affected and control farms was about 0.20 log10 cfu/ml. This constitutes a very large

decrease (2.5 million cfu/ml).

The coliform counts for all 11 houses were relatively low ranging from 0.10 to

3.88 log10 cfu/ml. This is consistent with previous studies (Hartel et al, 2000; Martin

et al, 1998; Vizzier-Thaxton et al, 2003). Hartel et al (2000) reported that 10 out of

the 20 fresh litter samples collected had levels lower than one log10 cfu/g. Martin et al

(1998) likewise reported that five out of 86 litter samples collected all across Georgia

had detectable coliform counts. Vizzier-Thaxton et al (2003) also reported average

litter coliform counts in Mississippi to be one log10 cfu/g. In contrast, Terzich et al

(2000)’s multi-state survey reported coliform counts ranging from 6 to 8 log10 cfu/g.

This discrepancy cannot be explained. These varying observations may be attributed

to the different geographical locations of these studies.

15

0123456789

A B C D E F G H I J K

Farms

Bact

eria

l cou

nts

(log

10 c

fu/m

l)

Total aerobic count coliform count Clostridium perfringens count

Figure 1. Average total aerobic, coliform and Clostridium perfringens counts in litter of gangrenous dermatitis-affected and control broiler farms * Farms A-H represent gangrenous dermatitis-affected farms * Farms I-K represent control farms

The Clostridium perfringens counts for both affected and control farms ranged

from 1 to 3 log10 cfu/ml or 2 to 4 log10 cfu/g. This is higher than the Omeira et al

(2006) study where the mean litter count for intensive broiler production was 1.05

log10 cfu/g. Although the difference in Clostridium perfringens counts between

gangrenous dermatitis affected farms and control farms was statistically significant,

the numerical or effective difference was only about 0.35 log10 cfu/ml or equivalent to

about 183 cfu/ml. This result suggests that gangrenous dermatitis affected farms may

not signify an effective increase in Clostridium perfringens counts compared to

control farms. If present, Clostridium perfringens could possibly pose a risk to food

safety along the production and processing sequence. This possibility is highlighted

by the study of Craven et al (2003) who were able to isolate the same ribotypes of

Clostridium perfringens in both fecal samples from grow-out farms and carcass rinses

from the associated processing plant. Considering that the infectious dose of

16

Clostridium perfringens type A is very high (McClane, 2001) and that there are many

more succeeding microbial reduction processing interventions, this slight increase in

counts from gangrenous dermatitis affected farms may not entail enough added Cl.

perfringens levels to pose an increased risk of Cl. perfringens type A food poisoning.

Also, it should be noted that only CPE (Cl. perfringens enterotoxin) positive isolates

can cause foodborne Cl. perfringens type A food poisoning (McClane, 2001).

17

Chapter 3: An Evaluation of Poultry Litter Treatment® (PLT®) on Clostridium spp. Recovery from Poultry Litter

3.1 Review of Literature

3.1.1 Clostridial Diseases in Poultry

Clostridium spp. are large, gram positive, sporeforming, anaerobic, rod-

shaped bacteria. In an incidence study, Craven et al (2001) detected Clostridium

perfringens in 94% of the flocks tested. Because Clostridium perfringens is

sporogenic and ubiquitous in soil due to its anaerobic nature, the study of the effects

of litter amendments to Clostridium spp. levels in poultry litter is important. It has

also been observed that litter clean-out delays recurrence of Clostridium outbreaks in

the poultry houses by 2-3 grow-out cycles (Bautista, personal communication).

Clostridium spp. infections in poultry are associated with four distinct diseases

namely: ulcerative enteritis, necrotic enteritis, gangrenous dermatitis, and botulism.

Ulcerative enteritis/Quail disease, caused by Clostridium colinum, is an acute

infection mainly seen in young poultry (Wages, 2003). It occurs in chickens at 4-12

weeks of age happening often with a co-infection or stress condition and producing

ambiguous signs such as watery white droppings (Wages, 2003). The spores

produced by the organism in the litter would be a permanent contamination (Wages,

2003).

Necrotic enteritis is caused by alpha and beta toxins produced by Clostridium

perfringens types A and C which are ubiquitous and part of the normal flora of

healthy chickens (Wages and Opengart, 2003b). Contaminated feed and litter have

18

been implicated in most outbreaks (Wages and Opengart, 2003b). Ambiguous signs

such as depression, anorexia, diarrhea, ruffled feathers, and sudden increase in

mortality are present (Wages and Opengart, 2003b).

Botulism/Limberneck/Western duck sickness is caused by the exotoxin of

Clostridium botulinum types A, C and E causing flaccid paralysis of the legs, wings,

neck, and eyelids (Dohms, 2003). The source of infection involves maggots festering

in the gut of bird carcasses, small crustraceans, and insect larvae (Dohms, 2003).

Gangrenous dermatitis is caused by Clostridium septicum, Clostridium

perfringens type A, and Staphylococcus aureus infection. A more detailed review of

literature is discussed in Chapter 2.

3.1.2 Poultry Litter Amendment

Ammonia is produced by microbial decomposition of uric acid, the principal

nitrogenous waste product in avian species. Other substrates for ammonia production

are organic nitrogen from avian feces and ammonium from the decomposition of

spilled feeds (Shah et al, 2006). The interplay of high pH, ammonia production, and

ureolytic bacteria causes a vicious cycle of promoting the propagation of each factor

as highlighted by Blake and Hess (2001). According to Blake and Hess (2001),

increasing pH increases ammonia concentration. High pH also encourages uric acid

decomposition which would produce more ammonia. High pH promotes the maximal

activity of uricase, the enzyme responsible for uric acid breakdown to allatoin, which

subsequently will be converted to urea, and finally to ammonia by urease (Black and

Hess, 2001; Ritz et al, 2004). Finally, high pH allows ureolytic bacteria to thrive,

resulting in the use of uricase. Ammonia production is also favored in high

19

temperature and high moisture situations making brooder flocks more at risk (Shah et

al, 2006).

Adding to the problem is the continuous and prolonged use of the same

poultry litter in many successive grow-outs, sometimes reaching two years or even

longer, resulting in ammonia build-up. The high price of litter raw materials like

wood shavings, the ever decreasing land available for litter disposal, and

environmental considerations are among the reasons for the recycling of poultry litter

(Shah et al, 2006).

The detrimental effects of ammonia on poultry performance and health have

been described in a few review articles (Al Homidan et al, 2003; Ritz et al, 2004;

Quarles and Caveny, 1979). Beker et al (2004) reported that the gain to feed ratio of

broilers was reduced significantly at 60 ppm ammonia (NH3) considering that

commercial poultry are usually exposed to 50 ppm NH3. The obvious consequence of

this is decreased production and lower profits for the grower. Some authors have also

associated increasing NH3 levels to increasing susceptibility to certain infections

(Moum et al, 1969) possibly due to stress, as well as to decreasing vaccination

efficacy (Kling and Quarles, 1974; Caveny et al, 1981). Anderson (1964) showed

higher infection rate for NDV for chickens exposed to 20 and 50 ppm ammonia than

control chickens.

Aside from being detrimental to poultry health, ammonia produced in broiler

houses is also an environmental and human health concern (Ritz et al, 2004). It helps

form fine particulate matters which can aggravate asthma (Shah et al, 2006).

20

There are five types of litter amendments that control ammonia: acidifiers,

alkaline materials, adsorbers, inhibitors, and microbial-enzymatic treatment. Among

the currently available litter acidifiers used in the industry are: Poultry Litter

Treatment/PLT® (93% NaHSO4), Poultry Guard®/Acidified clay (36% H2SO4 soaked

in clay) and Al+Clear®/Alum [Al2(SO4)3•14H2O] (Shah et al, 2006). PLT® is made

up of 93.2 % sodium hydrogen sulfate and 6.5 % sodium sulfate (Terzich et al,

1998b). PLT® acts by promoting ammonium retention by preventing it from being

converted to ammonia, thus lowering the litter pH. Free ammonium ions are

converted to ammonium sulfate, and the sodium binds with phosphate, forming

sodium phosphate (Terzich et al, 1998b). It also acts on the ureolytic bacteria as well

as the urease itself that these bacteria produce. Among the benefits given by PLT are:

decreased ammonia levels, decreased fuel usage due to the reduced need for

ventilation and heating during winter, improved flock performance and health,

reduced bacterial populations at the farm as well as in processing plant which is

mandated by HACCP, reduced beetle population, improved worker safety and health,

non-hazardous unlike Al+Clear®, and increased compliance with environmental and

public health regulations (Blake and Hess, 2001; Shah et al, 2006).

Terzich et al (2000) conducted a wide multi-state descriptive study to

determine the prevailing levels of bacteria in poultry litter. Clostridium perfringens

was not specifically counted, although gram positive counts were present in all states

tested. Vizzier Thaxton et al (2003), on the other hand, conducted a litter survey in

Mississippi. Although Clostridium perfringens was not specifically counted, general

anaerobic counts were shown to be constantly high.

21

There have been several studies evaluating the effect of PLT® on broiler

health (Terzich et al, 1998a; Terzich et al, 1998b; Nagaraj et al, 2007). Terzich et al

(1998a) reported that the ascites death rate in treated litter-raised broilers (5.9%) was

significantly lower than in untreated ones (31.5%). Terzich et al (1998b) also showed

that thoracic air sac and microscopic tracheal mucosal lesion scores in treated litter-

raised broilers were significantly lower than in untreated ones. On the other hand,

Nagaraj et al (2007) showed a decreased incidence of pododermatitis in treated litter-

raised broilers compared to untreated ones.

There have been studies evaluating the ability of PLT® to reduce the levels of

E. coli, Salmonella spp. and Campylobacter spp. (Line and Bailey, 2006; Line, 2002;

Payne et al, 2002; Pope and Cherry, 2000). However, some findings are conflicting.

Line (2002) reported that PLT® significantly reduced Campylobacter cecal

colonization frequency and levels but not Salmonella. On the other hand, Pope and

Cherry (2000) reported that Salmonella on farm carcass rinse counts were lower with

treatment but Campylobacter rinse counts were only marginally different between the

treated and control groups. Payne et al (2002) reported that artificially inoculated

litter had significantly lower Salmonella spp. counts in the treatment group. Line and

Bailey (2006) reported that PLT® delays the intestinal tract colonization by

Campylobacter but not Salmonella. These discrepancies might simply be due to

differences in experimental design and highlight the need for more studies.

22

3.2 Materials and Methods

3.2.1 Broiler House Layout and Design

An experimental broiler facility was divided into two separate houses, east

and west (Figure 2). Each house was further divided into five sampling locations

longitudinally: two near the side walls, one at the center of the house, and two under

the drinker lines (which are located between the center and the two side walls). These

five areas were then divided cross-sectionally into three blocks (near the door,

middle, and near the ventilation fan) (Figure 3). Each of the resulting cells (block X

location) was then divided again cross-sectionally into treatments: PLT-treated, salt-

treated, and control sub-cells organized in a randomized complete block design

(Figure 4). Salt was added as a treatment because it is the most common non-

commercial litter amendment used by poultry growers. PLT was applied at a rate of

100 lbs/ 1000 sq ft while salt was applied at a rate of 60 lbs per 1000 sq ft. Treatments

were applied over the soil pad. Six inch lengths of rebar were driven into the corners

of each sub-cell leaving about ¾ inch length protruding from the soil surface.

Three thousand chicks were placed in each house after taking the baseline

counts. The birds were vaccinated via drinking water with Newcastle Disease and

Infectious Bronchitis.

23

Experimental House LayoutExperimental House Layout

West East

Control RoomSilo Silo

Figure 2. Layout of the litter amendment experimental houses

Fan/SideMiddle/SideDoor/SideFan/WatererMiddle/WatererDoor/Waterer

Fan/CenterMiddle/CenterDoor/CenterFan/WatererMiddle/WatererDoor/Waterer

Fan/SideMiddle/SideDoor/Side

Door/SideDoor/WatererDoor/Center

Door/WatererDoor/Side

Middle/SideMiddle/WatererMiddle/CenterMiddle/Waterer

Middle/Side

Fan/SideFan/WatererFan/CenterFan/WatererFan/Side

East House

West House

Figure 3. Compartmentalization of the litter amendment experimental houses into blocks and location (Blocks/Location)

24

Experimental Broiler House LayoutExperimental Broiler House Layout

PLTPLT CC SS SS PLTPLT CC CC SS PLTPLT

CC SS PLTPLT PLTPLT CC SS SS PLTPLT CC

SS PLTPLT CC CC SS PLTPLT PLTPLT CC SS

PLTPLT CC SS SS PLTPLT CC CC SS PLTPLT

CC SS PLTPLT PLTPLT CC SS SS PLTPLT CC

Study design by: Dr. Susan White, Univ. of Delaware PLT=Poultry Litter Treatment; S=Salt; C=Control

Figure 2. Randomized complete block design of the litter amendment houses

3.2.2 Litter Sampling and Bacterial Enumeration

Each litter sample was collected by scooping a handful of litter and soil and

placing it in a sealable plastic bag during pre-treatment/baseline and at post-treatment

(weeks 3, 5, and 7). The samples were then transported for quantification to the

Maryland Department of Agriculture Animal Health Laboratory in College Park,

Maryland. Twenty-five grams of soil samples were dissolved in 225 ml PBS (Sigma-

Aldrich, St. Louis, MO) to make a 1:10 solution. The resulting solution was then

shaken for at least 15 minutes. Bacterial enumeration was performed by direct spread

plating using SFP (Becton, Dickenson and Company, Sparks, MD) agar for

Clostridium pefringens (Shahidi and Ferguson, 1971). Since it was shown in the pilot

study that most bacterial counts are within log 4, only three 1:10 dilutions were made.

The SFP agar culture plates were sealed in a plastic bag using a plastic sealer with an

25

AnaeroPak® (Mitsubishi Gas Company, Inc., New York, NY) included inside.

Bacterial enumeration was performed after 18-24 hours of incubation.

3.2.3 Statistical Analysis

All counts were converted into log forms. Using Statistical Analysis

System/SAS (SAS Institute Inc., Cary, NC), PROC GLM was employed to test

differences between blocks, locations, and treatments for statistical significance in

litter Clostridium perfringens counts during 3, 5, and 7 weeks of age as well as soil

pad Clostridium perfringens counts during pre-treatment/baseline and during harvest

using a randomized complete block design at α = 0.05. The t-test on the difference

between two treatment means was also employed at α = 0.05 to compare PLT counts

with Control counts and Salt counts with Control counts.

3.3 Results and Discussion Without factoring blocks and locations, the comparison of treatment effect on

Clostridium perfringens counts was inconclusive and did not show any general

pattern (Appendix C, Figures 5 and 6). At week 3 of age, PLT®-treated litter showed

the lowest average Clostridium perfringens count in the west house but almost the

same as the untreated litter in the east house. The salt-treated litter showed a lower

average count than the untreated litter in the west house but produced the highest

average count in the east house. At week 5 of age, PLT®-treated litter resulted in the

lowest average count in the east house but produced the highest average count in the

west house. The salt-treated litter resulted in a lower average count than the untreated

litter in the east house but produced almost the same average count in the west house.

26

At week 7 of age, the PLT®-treated litter showed a higher average count than the

untreated litter in the east house but almost the same average count in the west house.

The salt-treated litter, likewise, produced the same profile as the PLT®-treated litter.

During harvest (week 7), the PLT®-treated soil pad resulted in the lowest average

count in the east house but produced the highest average count in the west house. The

salt-treated soil pad resulted in a higher average count than the untreated soil pad in

the west but almost the same count in the east house. Using the t-test for comparison

between two means, all possible two treatment combinations produced statistically

insignificant differences (Appendix D).

0

0.5

1

1.5

2

2.5

Week 3, Litter Week 5, Litter Week 7, Litter Week 7, Soil

Time of sampling, sample taken

Clo

stri

dium

per

frin

gens

cou

nt

ControlPLT®NaCl

Figure 5. Average Clostridium perfringens counts in the east house

27

0

0.5

1

1.5

2

2.5

Week 3, Litter Week 5, Litter Week 7, Litter Week 7, Soil

Time of sampling, sample taken

Clo

stri

dium

per

frin

gens

cou

nt

ControlPLT®NaCl

Figure 6. Average Clostridium perfringens levels in the west house The results of factoring in the block and locations among the treatment in a

randomized complete design using GLM are shown in Appendix E and Appendix F.

The differences in the blocks and locations in the baseline soil counts for both houses

were all statistically insignificant. Only the differences between blocks for both

houses among the week 3 litter counts were statistically significant. In the east side,

the door block samples showed the highest least square means while in the west side,

the middle block showed the highest least square means. Both the differences

between the blocks and locations in the east house as well as the differences between

the blocks in the west house among the week 5 litter counts were statistically

significant. In the east side, the door block and waterer location samples showed the

highest least square means while in the west side, the fan block showed the highest

least square means. The statistically significant difference in the week 7 litter counts

28

were the same as those of week 5. In the east side, the door block and waterer

location samples showed the highest least square means while in the west side, the

middle block showed the highest least square means. Both the differences between

the locations and the interaction of the location and treatment in the east house as well

as the differences between the treatments in the west side among the harvest soil

counts were statistically significant. In the east side, the center location samples

showed the highest least square means, while in the west side, the salt treatment

showed the highest least square means and the PLT® treatment the lowest least square

means. These results show that for litter samples, blocks produced significantly

different results for both houses regardless of the timepoint of sampling. This

highlights the importance of using a randomized complete block design. For litter

samples collected beyond week 3, the locations in the east house produced

significantly different results. In both cases, the waterer location showed the highest

least square means. This can be explained by the fact that dripping water from

drinkers can enhance the growth of Clostridium perfringens. The results in the blocks

and locations are in contrast with the study of Craven et al (2001) who reported that

wall and fan swabs had the highest incidence of Clostridium perfringens. It should be

noted, however, that enumeration patterns in litter may not necessarily follow the

incidence pattern in area swabs. All the treatment differences except in the soil pad of

the west side during harvest were statistically insignificant after factoring the blocks

and location. In one case where the difference was statistically significant, PLT®-

treated soil pads showed lower least square means than the controls (Figure 7).

However, the effective difference was only 0.12 log10 cfu/ml or about 1.32 cfu/ml.

29

The prolonged five-month interval between treatment application and actual harvest

could possibly be the reason for this very small difference in counts.

0

0.1

0.2

0.3

0.4

0.5

0.6

Control PLT Salt

Treatment

Clo

stri

dium

per

frin

gens

cou

nt

Figure 7. Least square means of Clostridium perfringens counts in soil during harvest at the west house There have been several studies evaluating the effect of sodium bisulfate

treatment in litter on bacterial pathogen counts mostly dealing with coliform, E. coli,

Salmonella spp., and Campylobacter spp. (Pope and Cherry, 2000; Line, 2002; Payne

et al, 2002; Line and Bailey, 2006). Of these studies, only the Pope and Cherry

(2000) study directly measured the actual litter sample bacterial load to evaluate the

effect of the treatment. Line’s (2002) measure of effect was cecal sample and whole

carcass rinse counts while Payne et al (2002) evaluated the efficacy of sodium

bisulfate by measuring artificially inoculated litter in a pan. Clearly, the results from

the present study were not conclusive. One possible reason why this study failed is

that the design was flawed. There was no compartmentalization of the different

treatment groups. This would have caused the bacteria from litter of one treatment

group to be carried by broilers as they roamed around the experimental houses. In

30

previous PLT® evaluation studies, the researchers used a study design with separate

pens, chambers, or houses (Pope and Cherry, 2000; Nagaraj et al, 2003; Terzich et al,

1998a; Terzich et al, 1998b; Line 2002). Another major problem was the execution of

the study with regards to the length of time between the application of the treatments

(July 2007) and the placement of the birds (October 2007). The reason for this three

month delay was the need for fixing the electrical wiring for the lights of the

experimental house. One possible explanation is that the efficacy of the sodium

bisulfate was eroded by neutralization with ammonia being produced by remnant

ureolytic bacteria in the soil. In the Pope and Cherry (2000)’s study, the researchers

evaluated the litter only up to week 2 post-treatment with chick placement almost

immediately after treatment.

However, there are some valuable observations that can be taken from the

present study. It was shown that Clostridium perfringens counts in litter samples from

both houses were progressively declining over time (Figures 5 and 6). Whether this is

caused by increased competition by other bacteria due to increased bacterial load of

the houses as the broilers grew, or an effect of the sodium bisulfate and salt treatment

is impossible to ascertain with this design as other bacterial levels were not

quantified. This observation is similar to Craven et al’s (2001) observation where the

incidence of Clostridium perfringens from fecal samples inside the house decreased

with time. However, it should be noted that the enumeration pattern in litter may not

necessarily follow the incidence pattern in fecal samples collected over the litter. In

the soil pad during the harvest of broilers in both houses, where the effect of mixing

of bacteria by roaming broilers was not a factor, PLT®-treated litter had lower counts

31

than controls and salt-treated litter. This information is still useful since cumulative

bacterial load through successive litter use would eventually end up in the soil pads.

The reduction of bacterial load in the soil pad would be beneficial to eliminate

pathogen reservoirs when the litter is finally changed after several cycles. Even if

there is a barrier (the litter) between the soil pad and the broiler, successive litter use

over many grow-out cycles would lead to decreasing litter thickness. This scenario

would make it more likely that broilers can dig through the thin litter and have

contact with the soil pads. The different blocks at different time points for both

houses produced significantly different counts which shows that block design in this

study was very important.

Although HACCP is not currently mandated in the grow-out side of poultry

production, intervention strategies such as litter amendments may help lessen the

build-up of these foodborne pathogens in the poultry house environment and

subsequently reduce the level of exposure of broilers to these pathogens.

32

Chapter 4: Effect of Windrow Composting on the Microbiological Profile of Poultry Litter

4.1 Review of Literature

Poultry litter in the Delmarva peninsula is currently re-used for several cycles,

sometimes for as long as two years (Bautista, personal communication). Some of the

reasons why poultry litter is re-used are environmental concerns and the cost of new

litter materials (Lavergne et al, 2006). One of the other major uses of poultry litter is

as crop fertilizer. Direct applications of poultry litter as organic fertilizer has some

deleterious consequences such as potential spread of pathogens (Kelleher et al, 2002).

In addition to being used as a fertilizer, poultry litter is also sometimes used as an

alternative feedstuff for beef and dairy cattle (Martin et al, 1998; Terzich et al, 2000;

Jeffrey et al, 1998). Among the alternatives in disposing poultry litter are composting,

centralized anaerobic digestion, and direct combustion (Kelleher et al, 2002).

Composting may be a cheaper alternative than litter amendment in eliminating

bacterial pathogens in used litter (Macklin et al, 2006). Windrow pile heating or

windrow composting is a method used to sanitize recycled poultry litter. Composting

is not merely the piling of litter and manure to generate heat but is an active aerobic

process requiring aerobic bacteria, oxygen, and a carbon source to drive the

decomposition. There are several types of composts namely: static piles, passively

aerated piles, and mechanically turned windrows (Brodie et al, undated). According

to Brodie et al (undated), although static pile composting requires less labor, it does

take a longer time than turned compost to achieve the required degradation

33

temperature. In fact, mechanically turned windrow compost would only take only

about 5-10 days to complete (Macklin et al, 2007). Since poultry litter naturally

would have a very high nitrogen (N) content due to uric acid and partially digested

proteins, additional carbon (C) sources usually from organic material such as

sawdust, pine shavings, or peanut hull have to be added in order to achieve an

appropriate C:N ratio for optimum degradation (Atkinson et al, 1996). According to

Brodie et al (undated), the ideal C:N compost ratio is 25:1 to 35:1. The optimum

moisture content for composting is between 40% and 60% (Flory et al, 2006).

Compost that is too dry (<40% moisture) or too wet (>60% moisture) will not reach

the desired temperature of 1350 F (Flory et al, 2006). Too dry compost would reduce

the microbial diversity needed for efficient composting while too wet compost would

pose the risk of increasing ammonia volatization for the next flock (Lavergne et al,

2006). The study of Lavergne et al (2006) pegged the ideal moisture content at 32-

35%. In contrast, Brodie et al (undated) set the ideal moisture content much higher at

50-60%. The internal temperature of the compost must reach 1350 to 1450 F (Tablante

et al, 2002). If internal temperature drops to 1150 to 1250 F, the compost must be

turned for aeration (Tablante et al, 2002). According to Macklin et al (2007), there

are three mechanisms whereby pathogens are killed during composting namely: heat,

ammonia, and competition with fellow bacteria. There is a dearth of literature on

specifically evaluating the efficacy of windrow pile heating in reducing the bacterial

load of poultry litter. According to Wilkinson (2007), composting can control most

pathogens except spores and prions. Macklin et al (2006) showed reduction in both

aerobic and anaerobic bacterial counts after in-house static composting. Lavergne et

34

al (2006) showed that windrow composting can reduce anaerobic bacterial counts by

as much as 1.26 log10 cfu/g. Macklin et al (2008) showed reduction in aerobic,

anaerobic, Salmonella, Campylobacter, and Clostridium perfringens counts after in-

house windrow composting. However, both windrow studies by Lavergne et al

(2006) and Macklin et al (2008) did not involve turning/re-aeration. The objective of

this study was to evaluate the efficacy of windrow-composting in reducing the

bacterial load of poultry litter.

4.2 Materials and Methods

4.2.1 Windrow Construction and Litter Sampling

All poultry houses involved in this study had a history of gangrenous

dermatitis. The compost windrows consisted of poultry litter, wood chips, and

sawdust. No additional carbon source was added. All the feeders and drinkers were

raised to make room for the compost windrow and tractor equipment. All the

compacted and high moisture sublayer/cakes were removed using a skid steer loader.

The interior of the house was pressure-washed to remove dust buildup. A skid steer

loader or a tractor equipped with saw tooth paddle aerator (Brown Bear Corp., Iowa)

was used to construct the windrow. The windrow was about 10 ft wide and 5 ft high,

extending the whole length of the house. Depending on the size of the house and the

depth of the litter, two or three columns of windrows were constructed. The compost

was turned after 4.5 days, using the tractor equipped with a saw tooth paddle aerator.

Ten (10) to 60 litter samples were taken before the windrow was formed and after it

was spread. Sampling was performed by scooping a handful of litter and placing it in

35

a sealable plastic bag. The samples were then shipped overnight for quantification to

the Maryland Department of Agriculture Animal Health Laboratory in College Park,

Maryland.

4.2.2 Bacterial Enumeration

Twenty-five grams of litter samples were dissolved in 225 ml PBS (Sigma-

Aldrich, St. Louis, MO) to make a 1:10 solution. The resulting solution was then

shaken for at least 15 minutes. Bacterial quantification was performed by direct

spread plating using MacConkey agar (Becton, Dickenson and Company, Sparks,

MD) for coliform count, Trypticase Soy agar (Becton, Dickenson and Company,

Sparks, MD) for total aerobic count, and Shahidi-Ferguson Perfringens agar (Becton,

Dickenson and Company, Sparks, MD) for Clostridium pefringens. Only three 1:10

dilutions were made. The SFP agar (Shahidi and Ferguson, 1971) culture plates were

sealed in a plastic bag using a commercial kitchen food sealer with an AnaeroPak®

(Mitsubishi Gas Company, Inc., New York, NY) included inside. Quantification was

performed after 20-24 hours of incubation. For the Salmonella isolation, 25 grams of

litter sample were dissolved in 225 ml of buffered peptone water (Becton, Dickenson

and Company, Sparks, MD). This solution was allowed to incubate at 370C for 24

hours. Then, 1 ml from this pre-enrichment solution was added to 9 ml of selective

enrichment, tetrathionate broth (Becton, Dickenson and Company, Sparks, MD). The

broth culture was allowed to incubate at 420C for 24 hours. Thereafter, 1 ml from this

broth culture was transferred to a fresh 9 ml tetrathionate broth. This solution was

allowed to undergo delayed enrichment-recovery for 5-7 days at room temperature.

After delayed enrichment, a loopful of the broth culture was streaked onto an

36

XLT4/Xylose Lysine Tergitol-4 (Becton, Dickenson and Company, Sparks, MD)

selective plate in a four-quadrant streaking fashion. Suspected typical colonies (black

with pink periphery) were tested biochemically using TSI/Triple Sugar Iron (Becton,

Dickenson and Company, Sparks, MD) and LIA/Lysine Iron agar (Becton, Dickenson

and Company, Sparks, MD) tubes. All colonies producing typical Salmonella profile

in TSI and LIA tubes were confirmed using the API20E® Enteric Identification

System (BioMerieux, Inc, Hazelwood, MO).

4.2.3 Statistical Analysis

All counts were converted into log form. Using Statistical Analysis

System/SAS (SAS Institute Inc., Cary, NC), the difference in mean bacterial count

between pre-windrow and post-windrow compost was tested for significance using

the t-test at α = 0.05.

4.3 Results and Discussion A total of four houses were evaluated for bacterial load reduction before and

after windrow-composting. Two houses showed a statistically significant reduction in

total aerobic count, coliform count, and Salmonella spp incidence (Appendix G,

Appendix H. and Figure 8). All four houses showed a numerical reduction in

Clostridium perfringens counts. However, only three of these reductions were

statistically significant.

37

012345678

House A,Pre-

compost

House A,Post-

compost

House B,Pre-

compost

House B,Post-

compost

House C,Pre-

compost

House C,Post-

compost

House D,Pre-

compost

House D,Post-

compost

Houses, Time of sampling

Bac

teria

l cou

nts

(log

10 c

fu/m

l)

Total aerobic count Coliform count Clostridium perfringens count

*

***

*

***

*

***

*

***

*

**

*

**

*

*****

*

*****

Figure 8. Average total aerobic, coliform and Clostridium perfringens counts in litter pre- and post-composting * indicates that the total aerobic count reduction is statistically significant **indicates that the coliform count reduction is statistically significant ***indicates that the Clostridium perfringens count reduction is statistically significant Two of the houses showed a significant increase in TAC after windrowing.

This may be attributed to the delay in counting. The other two houses showed a

reduction in TAC of about 1 to 2 log10 cfu/ml. This is consistent with the studies of

Haque and Vandepopuliere (1994) and Macklin et al (2006). Haque and

Vandepopuliere (1994) showed a similar reduction after 22 days of composting.

Macklin et al (2006) showed aerobic bacterial reduction of about 1 log10 cfu/ml both

in the external and internal areas of the in-house compost pile after just two weeks.

Two houses showed undetectable coliform counts both before and after

windrow formation, making comparison impossible. This is consistent with previous

studies that have shown that coliform counts in litter are inherently minute (Hartel et

38

al, 2000; Martin et al, 1998; Vizzier-Thaxton et al, 2003). Of the two houses where

there were pre-windrow coliform levels, both had coliform counts reduced to

undetectable levels after windrowing. This is similar with the study of Haque and

Vandepopuliere (1994) who reported that E. coli was undetectable after 22 days of

composting. This is also consistent with previous studies where four out of 52

composted poultry litter samples tested yielded E. coli (Jeffrey et al, 1998). This

pattern is similar to the findings of Macklin et al (2008) where the E. coli counts

decreased by as much as 2.274 log10 cfu/g after windrow composting.

Of the four houses where there were numerical reductions in the level of Cl.

perfringens, house D produced the largest reduction of more than 1 log10 cfu/ml.

Macklin et al (2006) showed anaerobic bacterial reduction of 2 log10 cfu/g both in the

top external and internal areas of in-house litter compost within just two weeks. This

result is comparable with the house D result which had a reduction in its Clostridium

perfringens count of about 1.25 log10 cfu/ml or about 2.25 log10 cfu/g. Macklin et al

(2008), on the other hand, showed 8.92 log10 cfu/g in Cl. perfringens count. This

large discrepancy may be explained because Macklin et al (2008) used artificially

high amounts of inoculated Cl. perfringens in their study. Houses A and B, the other

two houses which showed a significant reduction in Clostridium perfringens count,

had a Clostridium perfringens reduction of 0.78 and 0.63 log10 cfu/ml or about 1.78

and 1.63 log10 cfu/g, respectively. This is consistent with the study of Lavergne et al

(2006) which showed about 1.26 log10 cfu/g in anaerobic bacterial count. One house

(house C) showed an effective but not statistically significant reduction in Cl.

perfringens counts.

39

Two houses showed zero Salmonella spp. isolation both before and after

windrow formation, making comparison impossible. Again, this result is consistent

with the study of Haque and Vandepopuliere (1994) where Salmonella spp. was not

detected both before and after windrow formation. For the two houses that showed

Salmonella spp. isolation, one house resulted in a reduction of 30 percentage points

(from 40% to 10%) while the other resulted in a reduction of 17 percentage points

(from 17% to 0%). This is consistent with the study of Macklin et al (2008) which

showed undetectable Salmonella counts after windrow composting.

The present study shows that windrow-composting is an effective tool for

pathogen reduction in used poultry litter. Some authors like Martin et al (1998)

contend that there is no consistent effect of composting versus non-composting on

microbial numbers. However, they did not show any data to support this assumption.

Reduction of litter bacterial counts is very important in integrated poultry production,

especially when litter is recycled. Although HACCP is not currently mandated in the

tgrow-out side of poultry production, intervention strategies such as litter composting

may help lessen the build-up of these foodborne pathogens in the grow-out

environment and the minimize the subsequent exposure of broilers to these

pathogens. A good justification for this intervention strategy was suggested by

Corrier et al (1999) who found that Salmonella spp. incidence in the crops of

chickens after feed withdrawal increased instead of decreased as would normally be

expected. They suggested that broilers might have ingested Salmonella-infected litter

while being subjected to feed withdrawal. This increased incidence of Salmonella

spp. in the crop may be a source of Salmonella later on during processing, especially

40

during evisceration due to accidental nicking or squeezing of visceral organs by an

improperly aligned machine. Therefore, intervention strategies in litter management

to reduce the level of pathogens have a bearing even in the later steps of live

production.

41

Chapter 5: A Baseline Study of the Level of Bacterial Foodborne Pathogens at Different Stages of Poultry

Processing

5.1 Review of Literature

Poultry processing includes all the steps involved in transforming a live bird

into a ready to cook product. Wabeck (2002b) further subdivided poultry processing

into the so-called “first processing” from receiving to chilling and “second

processing” from chilling to shipping. This chapter will focus on the “first

processing” stage of poultry processing. The basic steps in “first processing” are: feed

withdrawal, catching and transport, holding, receiving, live-hanging, scalding,

defeathering (picking), evisceration, washing, reprocessing, and chilling.

42

Unloading

Scalder

Live HangStunner

Kill

PickerSinger

Hock Cutter

ChillerPre-chiller

IOBW

InspectionRehang

Venter

Eviscerator

Giblet HarvesterLung/Kidney RemovalCropper

Blood Tunnel

Rehang

Diagram adapted from: Keener et al (2004)

Figure 9. Basic layout of a poultry processing plant

The rationale for feed withdrawal is to reduce fecal contamination to the

poultry’s exterior during transport and holding and to the carcass from severed

viscera during processing, facilitated by increased clearance time of feces (Keener et

al, 2004). A carcass with filled ingesta could lead to feces coming out of the vent by

slight swinging and jerking of the processing line (Russell, 2002). According to

Wabeck (2002b), the optimum time for feed withdrawal is in the range of 8 to 12

hours before the start of actual processing because prolonged withdrawal time will

cause increased carcass shrinkage, watery feces, and weakened intestine and gall

bladder tensile strength which could lead to breakage during evisceration (Wabeck,

2002b; Russell, 2002). However, in studies by Ramirez et al (1997) and Corrier et al

(1999), feed withdrawal even increased the isolation rate of Salmonella in the crop

compared to the isolation rate before feed withdrawal. The explanation given by

43

Corrier et al (1999) was that feed deprivation could force broilers to eat Salmonella

contaminated litter. In a similar study by Byrd et al (1998), feed withdrawal also

increased the isolation rate of Campylobacter in the crop as compared to the isolation

rate before feed withdrawal. Although, Northcutt et al (2003a) reported that even

though Campylobacter counts may increase in the pre-chill carcass when subjected to

12 hours feed withdrawal, counts after chilling are not affected by feed withdrawal.

One intervention strategy suggested in the study by Hinton et al (2000) was feeding a

glucose-based cocktail supplement during the feed withdrawal process which resulted

in fewer Salmonella recovery due to competitive exclusion by lactic acid bacteria.

There are currently four types of live-hauling systems employed by poultry

integrators in the United States namely: steel compartmentalized cages, sliding

drawer system, plastic or wooden coops, and mechanized harvester (Wabeck, 2002b).

Mechanized harvesting seems to be promising as research has shown that it produces

less downgrade defects than other systems (Wabeck, 2002b). Transportation, being

stressful in nature, causes reduced intestinal immunity and hence, allows increased

colonization of intestinal bacteria (Keener et al, 2004). This was shown in an

experiment by Line et al (1997) who reported that Salmonella colonization rate after

artificial inoculation is five-fold higher in the ceca after transport than before

transport. In a study by Buhr et al (2000), pre-scalding carcasses hauled in solid

floorings had significantly higher counts of coliform and E. coli compared to those

hauled in wire mesh floorings. However, there was no significant difference in

coliform and E. coli counts between the two transport floorings if the carcasses were

taken after picking.

44

Although the studies of Northcutt et al (2003a) and Buhr et al (2000) indicate

that increased bacterial load upstream of the processing line will be offset by the

successive later steps such as the chiller, it is still important to make an effort to

reduce bacterial load in the initial stages. This is because excessively dirty birds

coming to the processing plant might overwhelm some processing equipment such as

the scalding and chilling tanks and cause cross contamination downstream (Russell,

2002).

Poultry-laden trucks wait an average of 5 hours in large holding sheds before

being unloaded (Wabeck, 2002b). The major consideration in the holding stage is not

contamination but losses due to heat stress. The time of receiving coincides with the

end of feed withdrawal and the start of actual processing. The birds are then hanged

by their shackles at the start the actual processing line. Red lighting, bars for rubbing

against the breast, and a minimum of about one minute interval from shackling to

stunning are facilitated in order to calm the poultry prior to slaughter (Wabeck,

2002b).

The purpose of stunning is to facilitate optimum killing-bleeding and feather

release. There are two methods of stunning: electrical and gas/chemical (Heath et al,

1994). According to the study done by Heath et al (1992), 85% of the 279 processing

plants surveyed all over the U.S. use electrical stunning while the rest operate without

stunning or use gas. The gas system is generally used outside the United States.

Stunning beyond 35 volts, 0.3 amps for 8 seconds may result in broken clavicles,

pericardial sac bleeding, and ruptured blood vessels in the breast muscles (Wabeck,

45

2002b). An interval of about 15 seconds is needed from stunning to killing to allow

slight relaxation of the muscle (Wabeck, 2002b).

The two methods of slaughter are either manual (as in Kosher) or mechanical.

According to Wabeck (2002b), the optimal way of mechanical slaughtering is by

cutting only one side of the neck to avoid severing the trachea but still being able to

cut both the jugular vein and carotid artery. The rationale for avoiding the trachea is

to ensure that the bird is still breathing, thereby enabling proper bleeding (Wabeck,

2002b). The carcass is then allowed to bleed by a minimum of one minute to ensure

that birds are no longer breathing before entering the scalding tank. Other than for

animal welfare purposes, the other reasons for avoiding the trachea are to prevent

contamination of the air sacs which happens if the birds are still breathing in the

scalder tank and to avoid blood from adding to the biological oxygen demand (BOD)

of the scald water draining to the waste system (Wabeck, 2002b).

The purpose of the scalder is to aid the defeathering process by increasing the

feather density and friction with the picker fingers (Wabeck, 2002b), by breaking

down the proteins that hold the feathers in place (Bennett, 2006), and by opening the

feather follicles (Keener et al, 2004). The scalding stage is a crucial step for cross

contamination because the opened feather follicles only close during the chilling

process, trapping pathogens in contaminated tank water within the feather follicles

(Keener et al, 2004). Because of the potential for cross contamination, several

intervention strategies have been implemented to reduce this risk. Among these

strategies are the use of a surge-agitation, counter-current tank, multi-stage tanks; a

pre-scald brush; a post-scald rinse; an overflow of one quart fresh water per bird;

46

monitoring scald water pH; and keeping the water temperature to optimum levels

(Wabeck, 2002b; Russell, 2002; Bennett, 2006). There are two methods of scalding:

immersion and steam-spraying (Bennett, 2006). There are also two types of scalding

based on the desired product: soft and hard scald (Wabeck, 2002b). A hard scald

employs higher temperature (59-640C) but shorter time (30-75 seconds) while a soft

scald employs lower temperature (51-540C) but longer time (90-120 seconds)

(Bennett, 2006). In a study of 3-tank, counterflow scalder, Cason et al (2000) were

able to show decreasing coliform and E. coli counts in scald water with each

succeeding tank. Even the percentage of Salmonella isolation rate decreased in tank 3

compared to the two previous tanks. In the comparative study between a counter-

current scalder (James et al, 1992b) and a conventional scalder (James et al, 1992a),

aerobic plate counts and Salmonella isolation rates from carcass rinses taken from the

counter-current-modified plant were significantly less than those from the

conventional baseline plant throughout the processing steps. In a comparative study

between a conventional multi-pass, one tank and a single pass, 3-tank, counter current

with a pre-scald rinse and post-scald washer by Waldroup et al (1993), total aerobic,

coliform, and E. coli counts as well as Salmonella incidence from carcass drips at

post-scald were all significantly lower in the modified 3-tank, counter-current tank

than in the conventional one. However, there was no significant difference in the

microbiological profile of carcass rinses between the two scalder types at the level of

post-evisceration and post-chill. In the comparative study of Cason et al (1999)

between a two-pass, single tank and a two-pass, three tank scalder, there was no

difference in aerobic bacterial counts in carcass rinses. However, it should be noted

47

that the carcass samples were taken after the defeathering stage, making it impossible

to accurately conclude that the two scalder types had the same performance. In a

study by Berrang and Dickens (2000), counts of total aerobic, coliform, E. coli, and

Campylobacter from carcass rinses all decreased significantly after scalding by 1.8,

2.1, 2.2 and 2.9 log10 cfu/ml, respectively. This is in agreement with the study by Izat

et al (1988) where all three processing plants had ≥ 1.84 log10 cfu/1000 cm2 reduction

in Campylobacter counts.

The actual defeathering process is accomplished by short, firm rubber fingers

with rippled surfaces called roughers or pickers (Wabeck, 2002b). The process is

refined further by passing through pinning (to manually remove the pin feathers),

singeing (to burn hair feathers) and rinse-washer (to wash off blood, feathers, and

loose epidermis) (Wabeck, 2002b). This stage of poultry processing is a critical

control point for cross contamination because the rubber fingers may transfer

pathogens to previously uncontaminated carcasses (Keener et al, 2004). In fact, in the

study of Berrang and Dickens (2000), coliform, E. coli and Campylobacter counts all

increased after the defeathering stage by 0.5, 0.7 and 1.9 log10 cfu/ml, respectively.

This is in congruence with the study of Izat et al (1988) who found an increase in

Campylobacter counts in three plants tested. Some of the control measures done to

address this problem are continuous rinsing of carcass and picking equipment and use

of 18-30 ppm chlorine rinse (Bennett, 2006). Head removal and hock cutting are done

just after the post-picker bird washer (Wabeck, 2002b).

Upon cutting the hock joint, carcasses drop to the so-called “rehang belt”

where they are led out of the slaughtering line and into the eviscerating line to be

48

manually rehanged (Wabeck, 2002b). It should be noted that automated rehang is also

available and is recommended over manual rehang because it reduces external surface

cross contamination (Bennett, 2006).

Although the term “eviscerate” may only refer to the scooping action by a

spoon or finger-type apparatus to withdraw the viscera, there are several different

processes that happen before and after this, constituting the whole “evisceration”

process. Among them are preen gland removal, venting, opening cut, presenting,

post-mortem inspection, trimming, reprocessing, salvage, giblet harvesting, lung

removal, crop removal, neck removal, and final inspection (Wabeck, 2002b). The

ultimate benefits of the feed withdrawal process are linked to the possible accidental

nicking of intestines during the evisceration step (Bennett, 2006). Russell (2002)

pointed out that maladjustment of the venter, opener, and eviscerator may cause cuts

in the intestine leading to fecal contamination of the carcasses. The risk of

contamination of carcasses is greater during evisceration because it involves

manipulation of the intestinal tract that naturally harbors pathogenic bacteria. Among

the control practices that Bennett (2006) has recommended are whole carcass rinse

with 20 ppm chlorine, harvesting relatively uniform size birds, enforcing proper

employee hygiene, extracting crops towards the direction of the head, and washing

equipment. In a comparative study between the Stork Gamco Nu-Tech Evisceration

System and the conventional streamlined inspection system, Russell and Walker

(1997) reported that one farm showed no change while another showed a significant

reduction in aerobic plate, coliform, and E. coli counts from rehang to cropper stages.

The Nu-Tech Evisceration System has an innovation of totally removing the viscera

49

from the carcass, hence eliminating the need for presenters (Russell and Walker,

1997). In the conventional streamlined inspection system, the viscera remain attached

to the carcass during inspection.

The purpose of the so-called “final washer” is to remove all the remaining

contaminants in the carcass such as blood, fecal materials, and tissue fragments like

membranes because of the zero tolerance policy of the USDA on fecal materials for

carcasses entering the chiller (Wabeck, 2002b, Rasekh et al, 2005). To avoid any

confusion, it should be noted that the role of the final washer has changed slightly

through time. Previously, its main role was to remove visible fecal contamination as

the main online reprocessing tool (20 to 50 ppm chlorine). With the advent of

different stronger antimicrobials, the final washer’s role is more on removing the

organic load to prime the efficacy of the online reprocessing spray immediately after

the washer. According to Kemp et al (2000), the removal of serum exudates and

debris by the washer preconditions the carcass surface for the chemical action of the

online reprocessing antimicrobial. The risk of fecal contamination was demonstrated

by Smith et al (2007) who showed that an artificially cecal contaminated carcass

could have greater coliform, E. coli, and Campylobacter counts than controls. This

was again demonstrated by Jimenez et al (2003) who found a significantly higher E.

coli count in fecal contaminated carcasses after evisceration than in controls.

There are three types of carcass washers: brush washers, cabinet washers, and

inside-outside bird washers (IOBW) (Keener et al, 2004). Among the factors

affecting the efficiency of washers highlighted by Keener et al (2004) are number and

types of washers, water temperature and pressure, nozzle type and arrangement, flow

50

rate, line speed, and the sanitizing agent used. Yang et al (1998) gave a brief

description of how an IOBW works. The carcass is initially sprayed on the outside

with 9 nozzles, then, the inside with one nozzle and finally, the outside again with 9

nozzles. The inside spraying is actually two parts. First, the carcass is tilted

horizontally to ensure the chemicals reach the inside through the rear end. Secondly,

the carcass is tilted by 600 which would cause the chemical solution to flush out via

the neck cavity. The effectiveness of the final washer in reducing some pathogenic

bacteria has been shown to be significant in some studies while minimal in others. In

a study by Jimenez et al (2003), the enterobacteriaceae, E. coli, and coliform counts

in fecal contaminated carcasses passing through the IOBW were decreased by 9.7%,

7.9%, and 0%, respectively. In a study by May (1974) using a spray type washer, total

aerobic counts were reduced at an average of 56% from four plants using swabs taken

from an area at the back of the carcass. In the study by Izat et al (1988), all three

plants tested had reduced Campylobacter counts. In contrast, in a study by Bashor et

al (2004), Campylobacter counts in the post-washer were merely reduced by a range

of 0.26 to 0.66 log10 cfu/ml in the four plants studied compared to counts just before

the final washer. In a study by Northcutt et al (2003b), coliform and E. coli counts

were not significantly decreased by IOBW.

Currently, most plants use continuous online processing (COP) type of

reprocessing, employing various antimicrobial chemical solutions in order to comply

with the USDA zero tolerance policy for carcasses with visible fecal contamination

entering the chiller (Russell, 2007b). A more in-depth review of literature is discussed

in Chapter 6.

51

The main purpose of the chiller is to improve the quality and shelf life of the

carcass by limiting the growth of spoilage and pathogenic bacteria (Oyarzabal, 2005).

Chilling is not merely putting the carcass in a tank filled with slush ice as the USDA

mandates chillers to have an overflow rate of one-half gallon per bird chilled to

prevent microbial buildup (Wabeck, 2002b). The addition of 20 to 50 ppm chlorine

water is required by USDA to minimize cross contamination (Keener et al, 2004).

James et al (1992) was able to show that the reduction of aerobic plate and

Enterobacteriaceae counts in chillers with chlorination is significantly higher than in

chillers without chlorination. It is also important to maintain chiller water pH of 6 to

6.5 in order to favor the formation of hypochlorous acid over hypochlorite ion

(Russell and Keener, 2007; Bennett, 2006). Like the scalding tank, immersion chillers

also employ the countercurrent flow mechanism to prevent buildup of organic matter

(Sanchez et al, 2002; Russell, 2002). Chilling is actually made up of two stages: the

pre-chiller and final chiller. The purpose of the pre-chiller is to gradually lower the

temperature of the carcass to prevent tough meat caused by rapid muscle shortening

(Wabeck, 2002b). Pre-chillers are maintained at 500 to 650 F and consist mainly of the

overflow coming from the final chiller while the final chiller is maintained at 320 to

340 F (Wabeck, 2002b). The two main types of chilling are: immersion chilling and

air chilling. Air chilling is used in Europe and Canada where temperatures are

maintained in a refrigerated blast room to about 200 F (Sanchez et al, 2002; Wabeck,

2002b). The main advantage of immersion chilling is faster reduction of temperature

and the benefit of rinsing some bacterial load, while the main advantage of air

chilling is the prevention of cross contamination that occurs in the immersion chiller

52

tank (Sanchez et al, 2002). In a study by Sanchez et al (2002) comparing the

microbiological profile of carcasses between the two types of chillers, air chilled

carcasses were found to have 6% less incidence of Salmonella spp. and 9.4% less

incidence of Campylobacter spp. In the study of Berrang and Dickens (2000), total

aerobic, coliform, E. coli, and Campylobacter counts decreased by 0.7, 0.3, 0.4 and

0.8 log10 cfu/ml, respectively after chilling.

The final intervention strategies applied to poultry carcasses during processing

is the post-chill dip (Russell, 2007c) or a post-chill spray (Stopforth et al, 2007).

These interventions may be important if samples are recontaminated in the chiller

tank in underperforming plants (Stopforth et al, 2007). In a survey conducted by

Russell (2007c), acidified sodium chlorite is the most commonly used antimicrobial

(67 percent). In a study by Oyarzabal et al (2004), the use of acidified sodium

chlorite as a post-chill antimicrobial tremendously decreased Campylobacter counts

and prevalence and eliminated E. coli counts to undetectable levels in two

independent experiments.

5.2 Materials and Methods

5.2.1 Sample Collection

Carcass rinse samples were taken from a poultry processing plant in Arkansas.

Five samples were taken randomly from different stages of the processing line

namely: receiving, post-scalder, post-picker, rehang, post-evisceration, post-washer,

pre-SANOVATM, post-SANOVATM, and post-chill. All the samples taken were from

the same poultry flock. In order to ensure unbiased results, the first sample was taken

53

after some time that the processing line had been running. The sampling took place

early in the morning. Each whole carcass was placed in a bag of 100 ml buffered

peptone (Becton, Dickenson and Company, Sparks, MD) and shaken manually in the

so called “shake and bake” method for one minute. Fifty ml of the carcass rinse from

each bag were then transferred to a sterile dilution bottle and placed in a styrofoam

box containing crushed ice. Samples were held in this manner during transport.

Samples were then sent to the University of Delaware Lasher Poultry Diagnostic

Laboratory in Georgetown, Delaware.

5.2.2 Bacterial Enumeration.

Upon receiving the samples, serial dilutions were done up to 1:100,000. Direct

spread plating on MacConkey/MAC (Becton, Dickenson and Company, Sparks,

MD), Xylose Lysine Tergitol-4/XLT4 (Becton, Dickenson and Company, Sparks,

MD) and mCampy-Cefex (Oyarzabal et al, 2005) were used to quantify coliform,

Salmonella spp., and Campylobacter spp., respectively. A replicate was done for each

sample. mCampy-Cefex plates were sealed using a plastic sealer along with

MicroAeroPak™ (Mitsubishi Gas Company, Inc., New York, NY) which provided

the microaerophilic environment that Campylobacter spp. requires (Oyarzabal et al,

2005). Plates for coliform and Campylobacter spp. were read after 24 hrs incubation

while Salmonella spp. were read after 48 hrs incubation. MAC and XLT4 were

incubated at 37 0C while mCampy-Cefex was incubated at 42 0C. Five representative

colonies from each processing stage were taken from mCampy-Cefex plates. Their

biochemical profiles were tested to categorize them into either C. jejuni, C. coli, or C.

lari (Hunt et al, 1998).

54

5.2.3 Statistical Analysis

All bacterial counts of zero were replaced with 1 to allow log transformation.

All statistical measures were done in log10 cfu/ml unit. Using Statistical Analysis

System/SAS (SAS Institute Inc., Cary, NC), the differences in bacterial counts for

each pair of consecutive processing points were tested for statistical significance

using the t-test between two means at α=0.05.

5.3 Results and Discussion

There have been many publications evaluating the baseline total aerobic,

coliform, enterobacteriaceae. and E. coli levels as well as Salmonella spp. incidence

and levels in the different stages of the poultry processing plant (Appendix I and

Appendix J). A similar table for Campylobacter spp. counts was presented in a

review article by Keener et al (2004). Since baseline average coliform levels and

Salmonella isolation rates would depend on many different factors such as sampling

method (carcass rinse, carcass enrichment, neck skin, back swab, sponge swab), rinse

solution, neutralizing diluent, diluent volume, sample size, species sampled (chicken

or turkey), sampled plant specifications (e.g. with or without OLR), country/location

sampled, and sensitivity and specificity of method of microbiological enumeration or

isolation; it is not possible nor appropriate to compare the resultant bacterial levels to

the previous baseline study from other plants to determine whether process control is

properly being met in the current plant sampled. Rather, they may serve only as a

guide in evaluating the bacterial load reduction by process control. Under the HACCP

final rule set in 1996, only the post-chill carcass has a minimum performance

benchmark. The USDA-FSIS categorized generic E. coli counts into 3 classes: ≤100

55

cfu/ml (acceptable), >100 cfu/ml but ≤1000 cfu/ml (marginal), >1000 cfu/ml

(unacceptable) (FSIS, 1996b).

Coliform counts were shown to have decreased successively except after the

evisceration stage (Appendix K, Appendix L and Figure 10). The coliform levels

between the receiving and the post-scald carcass rinse decreased significantly by

about 0.95 log10 cfu/ml. This is consistent with the study of Berrang and Dickens

(2000) where the coliform counts decreased by 2.1 log10 cfu/ml. This huge

discrepancy in reduction might be due to the different pre-scalding points used

between the two studies. In our study, the pre-scald point was the receiving stage,

while in the Berrang and Dickens’ study, the pre-scald point was the post-bleed stage.

Blood could have caused an increase in the pre-scald (post-bleed) stage in the

Berrang and Dickens study as it is widely known that blood is a rich source of

nutrients for bacterial growth, causing the reduction to appear larger. This may also

be due to the difference in scalding specification. Berrang and Dickens used soft scald

(55.40C for 2.5 min) and a three stage counter-current tank while our study used hard

scald and only a single stage counter-current tank. This discrepancy between the two

studies caused by varying study design and plant specifications again highlights why

baseline data from other plants from previous studies cannot be fairly compared to the

data from the current plant being studied. The current hard immersion scalder

employs 610C water with 20 to 50 ppm chlorine for 90 seconds. Since the FSIS only

has performance benchmarks for post-chill carcass, we decided to compare our data

with that of post-scald coliform counts of previous studies. Previous post-scald

carcass rinses ranged from 1.8 to 2.9 log10 cfu/ml (Figure 11). This range is

56

considerably lower than our data of 4.53 log10 cfu/ml. This huge discrepancy cannot

be explained as the specific parameters and practices of the current plant under study

were not fully disclosed. The combined effects of high temperature and chlorine can

explain why there was almost a one log10 cfu/ml reduction in coliform counts.

Considering that the plant under study employs only a single stage tank, no pre-scald

brush and no post-scald rinse, the scalder tank performance and maintenance appear

to be excellent.

Coliform counts in a poultry processing plant

0.001.002.003.004.005.006.00

Receiv

ingPos

t-sca

ldPos

t-pick

Rehan

gPos

t-evis

cerat

ionPos

t-IOBW

Pre-SANOVATM

Post-S

ANOVATMPos

t-chil

l

Processing site

log1

0 cf

u/m

l **

**

*

Figure 10. Line chart of average coliform counts of carcass rinses taken from different stages of a poultry processing plant * indicates that the reduction is statistically significant

57

4.675 5.09

4.45 4.57

2.92.56

1.8 1.91

4.534.57

2.11.65

0

1

2

3

4

5

6

Russell (2005) Berrang andDickens (2000)

Waldroup et al(1993)a

Waldroup et al(1993)b

Waldroup et al(1993)c

Tan et al (2008)

Authors

Bac

teri

al c

ount

(log

10 c

fu/m

l)

Total aerobic count Coliform count E.coli count

Figure 11. Post-scald carcass rinse total aerobic, coliform and E.coli counts shown in previous studies

The coliform levels between the post-scald and the post-pick carcass rinses

decreased significantly by about 0.88 log10 cfu/ml. This is consistent with the study of

Göksoy et al (2004) where the coliform counts decreased significantly by 0.23 and

0.74 log10 cfu/g of neck skin. This contradicts the result of the study of Berrang and

Dickens (2000) where the coliform counts increased significantly by 0.5 log10 cfu/ml

due to possible cross-contamination. Berrang and Dickens suggested two possible

reasons why coliform counts would increase after the defeathering process. First, the

rubber fingers might serve as a cross contaminating substrate transferring coliform

bacteria to previously low-count carcasses. Secondly, the fingers might cause a

jerking effect to the carcass, squeezing out fecal content and causing increased

contamination. Previous post-pick carcass rinses ranged from 2.8 to 3.4 log10 cfu/ml

(Figure 12). Unlike the post-scald data, this range is very near our post-pick coliform

count of 3.64 log10 cfu/ml. Several new interventions like the post-pick spray or the

so-called the “New York” spray have been implemented in order to counter possible

cross-contamination as part of the multiple-hurdle approach. However in the study of

58

Stopforth et al (2007), the coliform count was merely reduced by 0.2 log10 cfu/ml

which is statistically insignificant. The significant 0.88 log10 cfu/ml reduction after

defeathering in this study can be attributed to the continuous in-process and post-pick

washer employed by the plant under study.

5

4.1 3.95 4.1 4.13.4 3.25 2.99 3.2

2.8

3.64

2.8 3.02 2.8 2.82.5

0

1

2

3

4

5

6

Berrang andDickens (2000)

Buhr et al(2000)a

Buhr et al(2000)b

Stopforth et al(2007)a

Stopforth et al(2007)b

Tan et al (2008)

Authors

Bac

teri

al c

ount

s (lo

g10

cfu

/ml)

Total aerobic count Coliform count E.coli count

Figure 12. Post-pick carcass rinse total aerobic, coliform and E.coli counts shown in previous studies The coliform levels between the rehang and the post-evisceration carcass

rinses increased significantly by about 0.77 log10 cfu/ml. This is consistent with the

study of Göksoy et al (2004) where the coliform counts increased, but not

significantly, by 0.07 and 0.15 log10 cfu/g of neck skin. The evisceration step poses a

huge opportunity for cross-contamination from the intestinal content (a natural

reservoir for pathogenic microorganisms) to spill to the carcasses. New technology

like the Nu-Tech® system that totally separates the viscera with the carcass (Russell

and Walker, 1997) and added intervention like post-evisceration wash (Stopforth et

al, 2007) may help lessen and counter the possible cross-contamination during

evisceration. Automated rehang and the Nu-Tech® system were employed in the

59

current study. Previous post-evisceration carcass rinses ranged from 2.71 to 3.27 log10

cfu/ml (Figure 13). This range is slightly lower than our post-evisceration coliform

counts of 3.71 log10 cfu/ml.

3.81

4.54.11

4.394

3.27 3.12.71

3.03 3.13.71

3.08

2.22.47

2.82 2.7

00.5

11.5

22.5

33.5

44.5

5

Fluckey et al(2003)

Berrang andDickens (2000)

Waldroup et al(1993)a

Waldroup et al(1993)b

Stopforth et al(2007)

Tan et al (2008)

Authors

Bac

teri

al c

ount

(log

10 c

fu/m

l)

Total aerobic count Coliform count E.coli count

Figure 13. Post-evisceration carcass rinse total aerobic, coliform, and E.coli counts shown in previous studies The coliform levels between the post-evisceration and the post-IOBW carcass

rinses decreased, but not significantly, by about 0.64 log10 cfu/ml. This is consistent

with the study of Northcutt et al (2003b) and Jimenez et al (2003) where the coliform

count reductions were not significant and absent, respectively. However in the study

of study of Berrang and Dickens (2000) and Stopforth et al (2007), the coliform

counts decreased significantly. Among the factors affecting the efficiency of washers

highlighted by Keener et al (2004) are number and types of washers, water

temperature and pressure, nozzle type and arrangement, flow rate, line speed and the

sanitizing agent used. In this study, the IOBW was employed only for 15 seconds. A

longer washer-carcass contact time might produce a significant decrease in coliform

counts. Previous post-IOBW carcass rinses ranged from 2.2 to 3.1 log10 cfu/ml

60

(Figure 14). This range is about the same as our post-IOBW coliform counts of 3.07

log10 cfu/ml.

3.6 3.4

4.7

3.3 3.6

2.2 2.43.1

2.2 2.53.07

1.5 1.45

2.82.1

2.7

1.9 2

00.5

11.5

22.5

33.5

44.5

5

Berrang andDickens(2000)

Oyarzabal etal (2004)a

Oyarzabal etal (2004)b

Northcutt etal (2003)a

Northcutt etal (2003)b

Stopforth etal (2007)a

Stopforth etal (2007)b

Tan et al(2008)

Authors

Bac

teri

al c

ount

(log

10 c

fu/m

l)

Total aerobic count Coliform count E.coli count

Figure 14. Post-IOBW carcass rinse total aerobic, coliform, and E.coli counts shown in previous studies

The coliform levels between the pre-SANOVATM and the post-

SANOVATMcarcass rinse decreased significantly by about 1.42 log10 cfu/ml. This is

consistent with several previous studies validating the tremendous effect of acidified

sodium chlorite in reducing bacterial load in broiler carcasses (Kemp et al, 2000;

Kemp et al, 2001; Oyarzabal et al, 2004; Stopforth et al, 2007). Using the worst case

scenario (all carcasses had fecal contamination), only two out of the 1,127 carcasses

tested failed to meet the USDA standard (Kemp et al, 2001). Kemp et al (2001)

showed an average of 2.28 log10 cfu/ml decrease in E. coli counts.

The coliform levels between the post- SANOVATM and the post-chill carcass

rinses decreased significantly by about 0.97 log10 cfu/ml. However, in the continuous

online processing study by Kemp et al (2001), the E. coli count increased

significantly by 0.25 log10 cfu/ml after chilling following the ASC (acidified sodium

chlorite/SANOVATM) treatment. They attributed this increase to the inability of the

ASC antimicrobial to penetrate areas below the carcass surface, allowing the residual

61

bacterial population to survive and come out by the tumbling action during chilling,

subsequently leading to a slight increase in counts. In the current study, the carcass

was allowed to stay in the chiller for 90 minutes at 340F with 20-50 ppm chlorine.

This prolonged contact time between chlorine treated chiller water with the carcass

may explain the significant reduction in coliform counts. Previous post-chill carcass

rinses ranged from 0.8 to 2.6 log10 cfu/ml (Figure 15). Our post-chill coliform counts,

0.38 log10 cfu/ml, were lower than this range.

00.5

11.5

22.5

33.5

44.5

Russell (2005) Oyarzabal etal (2004)a

Sanchez et al(2002)

Northcutt andothers

Berrang andDickens(2000)

James et al(1992a)

Waldroup et al(1993)a

Waldroup et al(1993)b

Bilgili et al(2002)

Stopforth et al(2007)a

Stopforth et al(2007)b

James et al(1992b)

Tan et al(2008)

Authors

Bac

teria

l cou

nt (l

og10

cfu

/ml)

Total aerobic count Coliform count E.coli count

Figure 15. Post-chill carcass rinse total aerobic, coliform and E.coli counts shown in previous studies

Salmonella counts were only present up until the post-pick stage. All three

sampling points only showed minimal Salmonella counts, not even reaching one log10

cfu/ml. Both the increase between the receiving and the post-scald and the reduction

between the post-scald and the post-pick in Salmonella counts were statistically

insignificant. These small counts can be attributed to the low sensitivity of the

technique used. According to Brichta-Harhay et al (2007), the detection limit of the

spiral plate count method in XLT4 using 50 µl in quadruplicates is only about five

cfu/ml or 0.70 log10 cfu/ml. In this study, direct selective agar plating in XLT4 was

done using 100 µl in duplicates. This detection limit is evident in the current study as

62

the highest average Salmonella spp. count was only 0.72 log10 cfu/ml. Direct selective

enrichment, in essence, has low sensitivity because of the lack of pre-enrichment. In a

comparative study between direct selective agar plating and pre-enrichment method

for isolation of Salmonella in eggs, the pre-enrichment technique consistently

produced higher isolation rates than the direct selective agar plating technique

(Valentín-Bon et al, 2003). The sampling technique (whole-carcass enrichment) by

Simmons et al (2003) who incubated buffered peptone water-soaked carcass for 24

hours at 370C before doing selective enrichment for incidence determination could be

applied to enumeration studies to yield better results. However, adding an enrichment

step poses a dilemma in enumeration studies because this could artificially inflate the

actual counts by allowing injured Salmonella to recover and multiply. Other

alternative methods in enumeration that can be used are real-time polymerase chain

reaction (RT-PCR) and most probable number (MPN) technique. However, RT-PCR

also presents some problems like low sensitivity and overestimation (Seo et al, 2006).

MPN, likewise, has some disadvantages because it requires too much time, labor, and

tubes (Seo et al, 2006). Most other Salmonella studies (Appendix J) in the processing

plant entail the use of incidence/isolation rate rather than enumeration because of the

difficulties cited. The American Society of Microbiology (ASM) has submitted a

comment to FSIS suggesting that enumeration rather than mere isolation be used in

evaluating pathogen reduction efficacy (Berkelman and Doyle, 2006). ASM contends

that there is no discrimination in efficacy of pathogen reduction with just positive

rates and without enumeration and that it is difficult to assess the efficacy of pathogen

reduction measures without a quantitative measurement of the organism. Because it

63

only takes one Salmonella cell to produce a positive result, one carcass with ten (one

log10 cfu/ml) Salmonella count and another with ten million (seven log10 cfu/ml)

Salmonella count would both produce the same weighted percentage positive. An

enumeration study may help identify or re-evaluate “critical control points” that could

lead to new or improved interventions or methods of pathogen reduction as well as

establish more accurate food safety standards. The ongoing new baseline study by the

FSIS is addressing this by adding Salmonella enumeration (FSIS, 2007a). However,

they will still be using the laborious and tedious MPN method. This poses a problem

in instituting MPN enumeration of Salmonella spp. for HACCP systems in processing

plants since chicken carcasses may well have been consumed already before the MPN

results are completed and reported. Until a fast and accurate enumeration method for

Salmonella spp. in carcass rinses can be employed, evaluation of Salmonella in

process control would likely be limited to isolation rates.

Campylobacter spp. counts were shown to have decreased successively except

after the chiller stage (Appendix K, Appendix L, and Figure 16). The Campylobacter

spp. levels between the receiving and the post-scald carcass rinse decreased

significantly by about 1.83 log10 cfu/ml. This is consistent with the study of Berrang

and Dickens (2000) where the Campylobacter spp. counts decreased by 2.9 log10

cfu/ml. This is also consistent with the study of Izat et al (1988) where

Campylobacter spp. counts decreased by about 1.84 to 2.48 log10 cfu/1000 cm2 in

three plants tested.

64

Campylobacter counts in a poultry processing plant

0123456

Receiv

ingPos

t-sca

ldPos

t-pick

Rehan

gPos

t-evis

cerat

ionPos

t-IOBW

Pre-SANOVATM

Post-S

ANOVATMPos

t-chil

l

Processing site

log 1

0 cfu

/ml *

**

*

Figure 16. Line chart of average Campylobacter spp. counts of carcass rinses taken from different stages of a poultry processing plant * indicates that the reduction is statistically significant

The Campylobacter spp. levels between the post-scald and the post-pick

carcass rinse decreased significantly by about 0.71 log10 cfu/ml. This does not follow

the result of the study of Berrang and Dickens (2000) and Izat et al (1988) where the

Campylobacter spp. counts even increased. In Berrang and Dickens (2000), the

counts increased significantly by 1.9 log10 cfu/ml. In Izat et al (1988), the counts

increased significantly in all three plants.

The Campylobacter spp. levels between the rehang and the post-evisceration

carcass rinses decreased significantly by about 0.49 log10 cfu/ml. This is consistent

with the study of Berrang and Dickens (2000) where the Campylobacter spp. counts

decreased, but not significantly, by 0.30 log10 cfu/ml. Izat et al (1988), on the other

hand, showed varying results where one plant had a significant increase, and the

second and third plants had an insignificant decrease.

65

Just like the coliform counts, the Campylobacter spp. levels between the post-

evisceration and the post-IOBW carcass rinses decreased, but not significantly, by

about 0.49 log10 cfu/ml. This is consistent with the study of Berrang and Dickens

(2000), Izat et al (1988), and Smith et al (2005) where there were variable

Campylobacter count reductions. Berrang and Dickens (2000) reported significant

reduction. Izat et al (1988) reported two plants having a significant reduction and one

plant having an insignificant reduction in Campylobacter counts. Smith et al (2005)

reported a reduction of about 1.8 log10 cfu/ml.

The Campylobacter spp. levels between the pre-SANOVATM and the post-

SANOVATM carcass rinses decreased significantly by about 1.12 log10 cfu/ml. This is

consistent with several previous studies validating the tremendous effect of acidified

sodium chlorite in reducing bacterial load in broiler carcasses (Kemp et al, 2001;

Oyarzabal et al, 2004; Bashor et al, 2004). Kemp et al (2001) showed an average of

2.56 log10 cfu/ml decrease in Campylobacter spp. counts. Bashor et al (2004), on the

other hand, showed a significant reduction of 1.26 log10 cfu/ml.

The Campylobacter spp. levels between the post- SANOVATM and the post-

chill carcass rinse increased, albeit not significantly, by about 0.20 log10 cfu/ml.

However, in the continuous online processing study by Kemp et al (2001), the

Campylobacter spp. count decreased significantly by 0.50 log10 cfu/ml after chilling

following the ASC (acidified sodium chlorite/SANOVATM) treatment.

It should be noted that a majority of the suspect colonies yielded negative

results in the biochemical test (18/25 to 25/25) (Appendix K). Several studies has

reported that Campy-Cefex agar to have a high level of contamination (false positive

66

colonies) (Line, 2001; Line et al, 2001). A more specific selective agar like the

Campy-Line and Campy-Line blood agar which have additional TTC

(Triphenyltetrazolium chloride) for increased resolution between the colonies and the

agar should have been employed instead (Line et al, 2001).

Although the Salmonella and Campylobacter results in this study were less

than ideal, the coliform counts did produce the intended goal of evaluating the

pathogen reduction efficacy of the different stages of the processing line. Scalding,

although expected to be a possible source of cross contamination, did show

significantly reduced coliform counts. It is unclear whether the same level of

reduction would be seen if the pre-scald point used is post-bleed rather than receiving.

The said processing plant does not employ a post-scald rinse but does add chlorine in

the scalding water. Picking, just like scalding, is another major concern for cross

contamination. Again, picking did show a significant reduction in coliform counts.

The said processing plant does employ continuous wash during defeathering and uses

a post-picking washer. Added attention must be given in the evisceration stage

because there was a statistically significant increase in coliform counts. Rehang in

this plant was reported to be automated. The Nu-Tech® evisceration system is

employed. The IOBW final wash did result in an effective reduction in this plant,

albeit not significant. The online reprocessing antimicrobial step resulted in the

largest reduction of almost 1.5 log10 cfu/ml. The chiller tank, just like the scald tank,

also presents the risk of cross contamination. This particular plant showed that even

the chiller significantly reduced coliform counts by almost one log10 cfu/ml. The final

average post-chill coliform count was 0.38 log10 cfu/ml. This is much lower than the

67

FSIS latest baseline generic E. coli limit for acceptable results for carcass rinse of

young chickens (35 cfu/ ml or about 1.54 log10 cfu/ml) (FSIS, 2005) and even much

lower than the HACCP established minimum performance benchmark of 100 cfu/ml

or two log10 cfu/ml for lower limit (FSIS, 1996b). The final average post-chill

Campylobacter spp. count was 0.61 log10 cfu/ml. This is almost equivalent to the

baseline data by Berrang et al (2007) for post-chill carcass rinse (0.43 log10 cfu/ml).

Based on the critical control point decision tree (FSIS, 1996b) and considering that

only the post-chill carcass rinse has a nationwide official baseline acceptable level,

we can only conclude that the chiller is indeed a critical control point. Furthermore,

this baseline study may serve as a control for future studies by this plant whenever

process control modification is implemented just like what was done by James et al

(1992a) and James et al (1992b).

68

Chapter 6: Comparison of Two Online Reprocessing (OLR) Antimicrobials

6.1 Review of Literature

In the past, the reprocessing rate in poultry processing plants has averaged

between 2 to 5 % (about 20,000 to 50,000 birds per week) (Fletcher et al, 1997).

Current authors estimate the reprocessing rate at 0.5 to 1 % (Russell, 2007a). Prior to

1989, carcasses that have accidentally been contaminated by feces during the

evisceration process were subjected to manual trimming in an off-line site (Russell,

2007a). This process, according to Russell (2007a), poses a lot of problems by virtue

of being labor intensive and triggers various probems associated with offline

processing such as: labor cost, labor issues (absenteeism), work-related injuries, and

the possibility of cross contamination (due to manually transporting the contaminated

carcass to an offline site and rehanging to the chiller). In addition, if the fecal

contamination is in the inside cavity, there would be no way to trim the carcass and

hence, it will be disposed of (Russell, 2007a). According to the rule published by the

USDA Food Safety and Quality Services in the Federal Register in 1978, procedures

such as trimming, vacuuming, washing or any combinations of such will be permitted

as reprocessing tools. In addition, if inner surfaces are reprocessed other than by

trimming, all the surfaces of the carcass must be treated with 20 ppm chlorine

solution (Rasekh et al, 2005). Initially, 20 ppm chlorine was instituted as a

reprocessing tool in an offline reprocessing station in accordance with the 1978 rule.

The USDA-FSIS allows up to 50 ppm chlorine in water for carcass washer

69

application and chiller water (Russell and Keener, 2007). According to a study by

Waldroup et al (1993a), 4 out of the 5 plants studied had carcasses with statistically

significant lower aerobic plate, coliform, and E.coli counts when reprocessed with 20

ppm chlorine compared to the inspection passed carcasses. Supporting the

justification for the use of online reprocessing, Fletcher et al (1997) undertook a

unique study that is usually quoted in current literature. Visually contaminated

carcasses after evisceration deemed by USDA line inspectors to be suitable for

reprocessing offline, were used as a criterion as “test” samples and were tested

against the control (those that passed the visual inspection after the chlorinated

IOBW). Fletcher et al (1997) showed that 67 % of carcasses that were being removed

by line inspectors, upon closer inspection, actually had no visual contamination and

could have been processed online. Adding those that have only a visual

contamination score of 1 (only 1 speck), which were found to be almost all

effectively eliminated by the IOBW in the control, a total of 81 % of carcasses pulled

for reprocessing would have been cleaned sufficiently later on by the IOBW if left in

the line. Furthermore, Fletcher et al (1997) showed that carcass processing using

online reprocessing had no difference in aerobic plate, coliform, and Campylobacter

counts compared to those that were manually reprocessed, alleviating initial

consumer concerns at that time that online reprocessing may not be as effective as

manual reprocessing. The development of continuous online reprocessing systems

soon followed with the use various new antimicrobial solutions developed other than

chlorine. Among these antimicrobial systems are: trisodium phosphate, acidified

sodium chlorite (SANOVATM), peracetic acid and combinations of peracetic acid and

70

octanoic acid (FMC 323® and Inspexx 100®), chlorine dioxide, acidified chlorine

(TomCo®), cetypyridinium chloride (Cecure®), mixture of acids (SteriFx® FreshFx®),

and acidified calcium sulfate (Safe2O®) (Russell, 2007a). Some other antimicrobial

products tested such as lactic acid and sodium bisulfate produced slight discoloration

of carcasses (Yang et al, 1998).

According to the survey undertaken by Russell (2007b) from 94 plants in the

US, acidified sodium chlorite (SANOVATM) by far is the most commonly used by

companies (33 percent). This is followed by trisodium phosphate (Rhodia®) (24%),

chlorine dioxide (15%), hypochlorous acid (9%), organic acids (6%), peracetic acid

(Perasafe®) (5%) and cetylpyridinium chloride and others making up the rest.

However, according to Rasekh et al (2005), 80 plants in the U.S. are currently using

TSP (trisodium phosphate) and 38 are using acidified sodium chloride.

Acidified sodium chlorite/ASC (Sanova Food Quality System/SANOVATM) is

a mixture of sodium chlorite (NaClO2) and a generally recognized as safe (GRAS)

organic acid which is usually citric acid. There have been several publications

assessing the efficacy of ASC in decreasing bacterial counts in poultry carcasses

when used as an antimicrobial agent for online reprocessing. Oxychlorous

intermediate is formed instantaneously when sodium chlorite and organic acid are

combined and come into contact with organic matter (Kemp et al, 2000). This level

increases when the solution decreases from pH 4 (Keener et al, 2004). The approved

dose for ASC is 500 to 1200 ppm to achieve a working pH of 2.3 to 2.9 in automated

reprocessing (Bennett, 2006). The mechanisms of action of ASC are oxidizing the

cell wall, attacking the sulfide and non-sulfide linkages in proteins, and non-

71

specifically attacking the amino acid of the cell membrane (Keener et al, 2004).

Kemp et al (2000) cited an unpublished report by Kemp (2000) that none of 10

microbes tested developed resistance after more than 100 divisions when placed in

sub-inhibitory dose of ASC. Kemp et al (2000) made a series of experiments to

evaluate different treatment parameters for ASC use. They found that ASC worked

better when carcasses are pre-washed than not, given at higher concentration

(1200ppm) than lower concentration (500 and 850 ppm) and applied by dipping than

spraying. There was no difference found when either citric acid or phosphoric acid

was used as the acidifier. The reason why citric acid is commonly used is because of

the apparent additional chelating activity it confers to the antimicrobial and the

concerns about disposing phosphate containing waste (Kemp et al, 2000) Kemp et al

(2000) also noted mild but transient skin whitening in the carcass at 1200 ppm that

was lost when subjected to the chiller. In a different study, Kemp et al (2001) showed

that combined reduction effects of final washer and ASC spray (continuous online

processing/COP) is much better than subjecting carcasses to offline reprocessing in

terms of E. coli and Campylobacter counts as well as Salmonella and Campylobacter

incidence rate. Considering all the samples used were initially visibly contaminated,

the COP was able to reduce E.coli by almost 2 logs and Campylobacter by about 1.5

logs more than offline reprocessing (Kemp et al, 2001). On the other hand, Bashor et

al (2004) reported that combined IOBW and ASC rinses decreased Campylobacter by

1.52 log cfu/ml. Kemp and Schneider (2000) showed that E. coli counts with

increasing ASC concentration in buffered peptone water (BPW) remained constant,

validating that BPW alone can neutralize the effect of ASC for enumeration purposes.

72

Perasafe® is composed of 15% peracetic acid and 10% hydrogen peroxide

(anonymous). The oxidizing activity of hydrogen peroxide causes disruption of the

cell membrane and protein synthesis through reaction with sulfhydryl, sulfide, amino

acid containing disulfide and nucleotide. In addition, the antimicrobial actions of

peracetic acid are acidifying the carcass surfaces and allowing the penetration of acids

into bacteria (Oyarzabal, 2005). Currently, there is no published literature on

assessing the use of Perasafe® as an online reprocessing antimicrobial. Since peracetic

acid may react with blood vessels, slight gray discoloration of the carcass may occur,

especially in highly vascular areas such as the neck (Russell, 2007b). In an

experiment by Dickens and Whittemore (1997) determining the separate effects of

acetic acid and hydrogen peroxide in microbial reduction, 1 % acetic acid was found

to have 0.6 log cfu/ml more reduction in total aerobic counts than controls while 3

different concentrations of hydrogen peroxide were found to have no increase in

reduction of total aerobic counts compared to controls. However, it should be noted

that the contact time for both antimicrobials is only 30 seconds. In a study by

Chantarapanont et al (2004), the GFP-Campylobacter jejuni count was decreased by

1.05 log cfu/ml when 100 ppm peracetic acid was applied for 15 minutes as a dip in

an attachment assay.

6.2 Materials and Methods

6.2.1 Sample Size and Collection

Ten carcass samples were taken daily for 8 days from both line 1 and line 2 at

three processing points namely: pre-OLR (online reprocessing), post-OLR, and post-

73

chill. Samples came from different flocks on different days of collection and were

collected simultaneously from line 1 and line 2.. Each whole carcass was placed in a

bag of 100 ml buffered peptone (Becton, Dickenson and Company, Sparks, MD) and

shaken manually in the so called “shake and bake” method for one minute. Fifty ml of

the carcass rinse from each bag were then placed in a cooler with ice packs and sent

to the Lasher Poultry Diagnostic Laboratory in Georgetown, Delaware.

6.2.2 Bacterial Enumeration

Upon receiving the samples, serial dilutions up to 1:100 were performed. One

ml of each sample dilution was inoculated into 3M® Petrifilm® Total Aerobic Count

Plate and Coliform Count Plate (3M Microbiology Products, St. Paul, MN) for total

aerobic and coliform counts, respectively. The cover film was then placed and the

inoculum was spread evenly on the Petrifilm® using a weight spreader. The films

were incubated at 37 0C for 24 hrs before counting (Russell, 2000).

6.2.3 Statistical Analysis

All bacterial counts of 0 were replaced with 1 to allow log transformation. All

statistical measures were done in log10 cfu/ml unit. Using Statistical Analysis

System/SAS (SAS Institute Inc., Cary, NC)’s PROC GLM (General Linear Model),

the absolute difference between bacterial counts for each pair of consecutive

processing points from one OLR antimicrobial agent were tested statistically against

that of the other OLR antimicrobial agent. This was calculated using with a contrast

formula: +1 -1 -1 +1. A GLM table between the Line, Trial (Day) and their

interaction was constructed using pre-OLR data.

74

6.3 Results and Discussion Both the two online reprocessing antimicrobials produced less than 1 log10

cfu/ml reduction for TAC after OLR treatment (Appendix M, Figure 17, and Figure

18). However, TAC counts were reduced by almost 2 log10 cfu/ml after chilling. This

observation seems to suggest a synergistic effect between the OLR antimicrobial and

the chlorine at the chill tank. This possible synergism was suggested by Oyarzabal et

al (2004). However, their experiment was in reverse of the current experiment where

acidified sodium chlorite dip was applied after chilling. These researchers suggested

that the chlorine and cold temperature during the chilling process had weakened the

bacterial cell wall, allowing acidified sodium chlorite to produce tremendous

reduction. It is unclear whether OLR or chilling, if applied first, would have a better

effect. A future comparative study is suggested. Coliform counts, likewise, were

reduced only by about 1 log10 cfu/ml post-OLR but about 2 log10 cfu/ml post-chill for

both antimicrobials (Appendix M, Figure 19, and Figure 20).

All differences of reduction in TAC and coliform counts between the two

antimicrobials were found to be statistically insignificant both in individual lines and

lines combined (Appendix N). The difference in counts between pre-OLR and post-

OLR, between the pre-OLR and post-chill, and between the post-OLR and post-chill

would represent the effect of the OLR antimicrobial, combined effects of OLR

antimicrobial and chlorine in the chill tank, and the chlorine in the chill tank,

respectively. The majority of these combinations suggest that Perasafe® has a slight

edge in bacterial reduction than SANOVATM, although this difference is statistically

insignificant. The main purpose of this study was to determine whether using a new

75

OLR antimicrobial (Perasafe®) would be more cost-effective than the currently used

antimicrobial (SANOVATM) Our study showed that there was no statistically

significant difference in performance between the two antimicrobials.

As a side study, the two lines were compared at the pre-OLR stage to

determine whether there was a difference in performance between the two lines with

regards to the evisceration and IOBW. There was a statistical difference between

lines in terms of coliform counts but not TAC (Appendix O). Line 1 showed lower

coliform counts than line 2 (Appendix P). In fact, it is obvious that Line 2 had higher

post-OLR coliform counts than Line 1 (Figure 19 and Figure 20). There were

statistically significant differences in both TAC and coliform counts in regards to day

of collection. Both TAC and coliform counts were lowest at day 2 of collection. Day

6 and day 1 resulted in the highest TAC and coliform counts, respectively. There was

a significant interaction between day of collection and line for coliform counts but not

for TAC counts. Line 1 at day 2 of collection showed the lowest coliform count while

line 2 of day 6 showed the highest coliform count.

76

00.5

11.5

22.5

33.5

44.5

5

Day 1,Line 1

Day 2,Line 1

Day 3,Line 1

Day 4,Line 1

Day 1,Line 2

Day 2,Line 2

Day 3,Line 2

Day 4,Line 2

Day sampled, Evisceration line

Tota

l aer

obic

cou

nt (l

og10

cfu

/ml)

Pre-OLRPost-OLRPost-chill

Figure 17. Average total aerobic counts (TAC) of carcass rinses taken from pre-OLR, post-OLR and post-chill stages of the processing plant using SANOVATM antimicrobial OLR=online reprocessing

0

1

2

3

4

5

6

Day 5,Line 1

Day 6,Line 1

Day 7,Line 1

Day 8,Line 1

Day 5,Line 2

Day 6,Line 2

Day 7,Line 2

Day 8,Line 2

Day sampled, Evisceration line

Tota

l aer

obic

cou

nt (l

og10

cfu

/ml)

Pre-OLRPost-OLRPost-chill

Figure 18. Average total aerobic counts (TAC) of carcass rinses taken from pre-OLR, post-OLR and post-chill stages of the processing plant using Perasafe® antimicrobial OLR=online reprocessing

77

0

0.5

1

1.5

22.5

3

3.5

4

4.5

Day 1,Line 1

Day 2,Line 1

Day 3,Line 1

Day 4,Line 1

Day 1,Line 2

Day 2,Line 2

Day 3,Line 2

Day 4,Line 2

Day sampled, Evisceration line

Colif

orm

cou

nt (l

og10

cfu

/ml)

Pre-OLRPost-OLRPost-chill

Figure 19. Average coliform counts of carcass rinses taken from pre-OLR, post-OLR and post-chill stages of the processing plant using SANOVATM antimicrobial OLR=online reprocessing

00.5

11.5

22.5

33.5

44.5

5

Day 5,Line 1

Day 6,Line 1

Day 7,Line 1

Day 8,Line 1

Day 5,Line 2

Day 6,Line 2

Day 7,Line 2

Day 8,Line 2

Day sampled, Evisceration line

Colif

orm

cou

nt (l

og10

cfu

/ml)

Pre-OLRPost-OLRPost-chill

Figure 20. Average coliform counts of carcass rinses taken from pre-OLR, post-OLR and post-chill stages of the processing plant using Perasafe® antimicrobial OLR=online reprocessing

78

Chapter 7: Summary and Conclusion

Although the Clostridium perfringens counts in gangrenous dermatitis

affected farms were statistically significantly higher than those in non-affected

(control) farms, the effective difference was only about 0.35 log10 cfu/ml or

equivalent to about 183 cfu/ml. The results of the comparative litter survey study

suggest that gangrenous dermatitis-affected farms may not signify an effective

increase in the level of the food-poisoning agent Clostridium perfringens type A.

Considering that previous studies have indicated that only about 5% of the global Cl.

perfringens isolates are positive to cpe gene (McClane, 2001), future survey studies

should include typing of isolates with this gene. Factoring the block and location

differences, only the soil pads during harvest (week 7) at the west house had a

statistically significant difference in Clostridium perfringens counts among treatments

with the PLT® sub-plot, resulting in lower counts than the controls (no treatment) and

the salt sub-plots. However, the effective difference in Cl. perfringens counts between

PLT® and control sub-plots was only about 0.12 log10 cfu/ml or about 1.32 cfu/ml.

Since the current grow-out practice is to re-use the litter for several grow-out cycles,

it is recommended that litter amendments also be applied to the soil pad as it might

serve as a reservoir of foodborne pathogens, particularly, Clostridium perfringens.

The litter amendment study showed that Clostridium perfringens counts in litter from

both houses were declining over time. In the windrow-composting studies, two

houses each showed a significant decrease in total aerobic and coliform counts as

79

well as in Salmonella spp. incidence. In addition, all four houses tested showed

numerical decreases in Clostridium perfringens counts. Although the Salmonella and

Campylobacter results of the processing line study were less than ideal, the coliform

counts did produce the intended goal of evaluating the pathogen reduction efficacy of

the different stages of the processing line. All processing steps except the evisceration

and IOBW resulted in a statistically significant reduction in coliform counts. All

processing steps except the IOBW and chiller resulted in a statistically significant

reduction in Campylobacter spp. counts. Since studies have reported that Campy-

Cefex agar has a high level of contamination (false positive colonies) (Line, 2001;

Line et al, 2001), a more specific selective agar like the Campy-Line and Campy-

Line blood agar is recommended for future processing plant baseline studies. In the

comparative OLR antimicrobial study, both TAC and coliform counts were reduced

only by about 1 log10 cfu/ml after the OLR but were reduced by 2 log10 cfu/ml after

the succeeding chiller, suggesting a synergistic effect between the OLR antimicrobial

and the chiller chlorine. It is suggested that a comparative study be done to compare

the effect when OLR is used before and after the chiller. The differences in reduction

between the two commercial antimicrobials were found to be statistically

insignificant. Line two of the comparative antimicrobial study showed a statistically

significant higher coliform count than line one suggesting that line 2 was more prone

to coliform contamination during the evisceration and IOBW stages of processing.

Similar in-house studies may be adopted by poultry integrators in their processing

plants to identify problem areas promptly in order to initiate timely corrective and

preventive actions. Finally, intervention strategies during farm production and

80

processing were found to be able to help reduce the level of common foodborne

pathogens.

81

Appendices

Appendix A. Average total aerobic, coliform and Clostridium perfringens counts in litter of gangrenous dermatitis-affected and control farms Total Aerobic

Counts (log10 cfu/ml±SE)

Coliform Counts (log10 cfu/ml±SE)

Clostridium perfringens Counts (log10 cfu/ml±SE)

Gangrenous Dermatitis-Affected Farms

Farm A (n=10) 6.57±0.14 0.10±0.10 3.00±0.14 Farm B (n=10) 5.81±0.17 1.18±0.50 2.15±0.10 Farm C (n=10) 6.49±0.22 1.23±0.38 2.80±0.13 Farm D (n=10) 7.41±0.17 1.12±0.34 2.15±0.17 Farm E (n=10) 7.30±0.21 1.83±0.37 2.07±0.18 Farm F (n=10) 7.83±0.10 2.29±0.33 2.27±0.08 Farm G (n=10) 5.81±0.16 1.06±0.45 3.04±0.16 Farm H (n=9) 7.58±0.20 3.88±0.31 2.67±0.13 Total (n=79) 6.84±0.10 1.56±0.17 2.52±0.06 Control Farms Farm I (n=10) 6.41±0.15 0.27±0.27 2.23±0.26 Farm J (n=10) 6.37±0.13 1.44±0.58 2.44±0.13 Farm K (n=10) 7.12±0.19 2.53±0.35 1.83±0.31 Total (n=30) 6.64±0.11 1.41±0.29 2.17±0.15

82

Appendix B. ANOVA table for total aerobic, coliform and Clostridium perfringens counts in litter of gangrenous dermatitis-affected and control farms Mean Square F-value P-value Interpretation Total Aerobic Counts Farm Status 0.99179239 3.45 0.0661 Not significant Farm (Farm Status)a 5.21042813 18.15 <0.0001 Significant Coliform Counts Farm Status 0.6566057 0.46 0.5014 Not significant Farm (Farm Status)a 12.0402945 8.35 <0.0001 Significant Cl. perfringens Counts Farm Status 2.700011400 8.75 0.0039 Significant Farm (Farm Status)a 1.148555864 4.81 <0.0001 Significant aFarm (Farm Status) row is the analysis of variance between all farms nested in their respective farm status category α = 0.05

83

Appendix C. Average Clostridium perfringens counts in soil and litter using PLT® (Sodium Bisulfate), salt and no treatments Control

(log10 cfu/ml±SE) East: n=15 West: n=15

PLT® (log10 cfu/ml±SE) East: n=15 West: n=15

Salt (log10 cfu/ml±SE) East: n=15 West: n=15

Litter, Week 3 East: 2.23±0.12 West: 2.22±0.14

East: 2.28±0.13 West: 2.03±0.10

East: 2.36±0.11 West: 2.05±0.15

Litter, Week 5 East: 1.63±0.13 West: 1.78±0.21

East: 1.53±0.22 West: 1.93±0.19

East: 1.57±0.17 West: 1.79±0.20

Litter, Week 7 East: 1.06±0.18 West: 1.19±0.21

East: 1.31±0.13 West: 1.21±0.18

East: 1.19±0.21 West: 1.16±0.18

Soil, Week 7 East: 0.50±0.13 West: 0.19±0.07

East: 0.34±0.11 West: 0.80±0.05

East: 0.56±0.15 West: 0.46±0.19

Each Cl. perfringens count is an average of duplicates.

84

Appendix D. Results of t-test between two treatment means of the litter amendment study Treatment

1 n=15

Treatment 2

N=15

Difference (log10

cfu/ml)

T-value

P-value

Interpretation

Control PLT® -0.0550 -0.31 0.7615 Not Significant

Control Salt -0.1310 -0.81 0.4230 Not Significant Litter, Week 3, East

PLT® Salt -0.0760 -0.44 0.6666 Not Significant Control PLT®

0.1908 1.12 0.2703 Not Significant Control Salt 0.1644 0.80 0.4332 Not Significant

Litter, Week 3, West

PLT® Salt -0.0260 -0.15 0.8848 Not Significant Control PLT®

0.0999 0.39 0.6969 Not Significant Control Salt 0.0563 0.26 0.7966 Not Significant

Litter, Week 5, East

PLT® Salt -0.0440 -0.16 0.8764 Not Significant Control PLT®

-0.1530 -0.55 0.5884 Not Significant Control Salt -0.0100 -0.03 0.9733 Not Significant

Litter, Week 5, West

PLT® Salt 0.1428 0.52 0.6060 Not Significant Control PLT®

-0.2510 -1.14 0.2649 Not Significant Control Salt -0.1250 -0.45 0.6537 Not Significant

Litter, Week 7, East

PLT® Salt 0.1264 0.52 0.6093 Not Significant Control PLT®

-0.0240 -0.09 0.9324 Not Significant Control Salt 0.0253 0.09 0.9271 Not Significant

Litter, Week 7, West

PLT® Salt 0.0491 0.19 0.8473 Not Significant Control PLT®

0.1573 0.90 0.3751 Not Significant Control Salt -0.0640 -0.32 0.7507 Not Significant

Soil, Week 7, East

PLT® Salt -0.2210 -1.19 0.2449 Not Significant Control PLT®

0.1121 1.33 0.1947 Not Significant Control Salt -0.2650 -1.32 0.1969 Not Significant

Soil, Week 7, West

PLT® Salt -0.3770 -1.95 0.0610 Not Significant α = 0.05

85

Appendix E. GLM table for Clostridium perfringens counts in litter and soil of the litter amendment study Source Mean

Square F-

value P-value Interpretation

Block 0.35102628 0.91 0.433 Not Significant Soil, Baseline, East

Location 0.99216300 2.57 0.125 Not significant

Block 1.05425566 3.06 0.091 Not significant Soil, Baseline, West

Location 0.54600500 1.59 0.252 Not significant

Block 1.78693089 12.42 <0.0001 Significant Location 0.36898466 2.56 0.091 Not significant Treatment 0.06901766 0.48 0.623 Not significant

Litter, Week 3, East

Location*Treatment 0.02806349 0.20 0.939 Not significant Block 1.32867156 5.98 0.005 Significant Location 0.20026610 0.90 0.415 Not significant Treatment 0.12755570 0.57 0.568 Not significant

Litter, Week 3, West

Location*Treatment 0.08570995 0.39 0.817 Not significant Block 3.35047128 14.49 <0.0001 Significant Location 2.56260871 11.08 0.0001 Significant Treatment 0.03865046 0.17 0.846 Not significant

Litter, Week 5, East

Location*Treatment 0.02377550 0.10 0.980 Not significant Block 6.40124711 21.73 <0.0001 Significant Location 0.46107862 1.57 0.223 Not significant Treatment 0.06976400 0.24 0.790 Not significant

Litter, Week 5, West

Location*Treatment 0.24758144 0.84 0.509 Not significant Block 3.47305835 13.40 <0.0001 Significant Location 1.01662431 3.92 0.029 Significant Treatment 0.30340765 1.17 0.322 Not significant

Litter, Week 7, East

Location*Treatment 0.38242228 1.47 0.231 Not significant Block 1.82546011 4.07 0.026 Significant Location 1.17803680 2.63 0.087 Not significant Treatment 0.11164873 0.25 0.781 Not significant

Litter, Week 7, West

Location*Treatment 0.36097762 0.80 0.530 Not significant Block 0.53332026 2.90 0.069 Not significant Location 0.79522848 4.32 0.021 Significant Treatment 0.48536326 2.63 0.086 Not significant

Soil, Week 7, East

Location*Treatment 0.50854518 2.76 0.043 Significant Block 0.03752777 0.19 0.831 Not significant Location 0.30673472 1.51 0.234 Not significant Treatment 0.73764568 3.64 0.036 Significant

Soil, Week 7, West

Location*Treatment 0.32694706 1.61 0.193 Not significant α = 0.05

86

Appendix F. Least square means of Clostridium perfringens counts in soil and litter based on block, location, and treatment Block log10

cfu/ml±SE Location log10

cfu/ml±SE Treatment log10

cfu/ml±SE Door 1.25±0.28 Center 1.84±0.36 Fan 1.73±0.28 Side 1.05±0.25

Soil, Baseline, East Middle 1.68±0.28 Waterer 1.76±0.25

Door 0.33±0.27 Center 1.24±0.34 Fan 0.80±0.27 Side 0.56±0.24

Soil, Baseline, West Middle 1.25±0.27 Waterer 0.58±0.24

Door 2.60±0.10 Center 2.36±0.13 Control 2.23±0.10 Fan 1.93±0.10 Side 2.14±0.09 PLT® 2.32±0.10

Litter, Week 3, East Middle 2.38±0.10 Waterer 2.41±0.09 Salt 2.37±0.10

Door 1.76±0.12 Center 2.12±0.16 Control 2.21±0.13 Fan 2.22±0.12 Side 1.99±0.11 PLT® 2.04±0.13

Litter, Week 3, West Middle 2.32±0.12 Waterer 2.20±0.11 Salt 2.05±0.13

Door 1.98±0.13 Center 1.48±0.16 Control 1.62±0.13 Fan 1.05±0.13 Side 1.23±0.11 PLT® 1.52±0.13

Litter, Week 5, East Middle 1.65±0.13 Waterer 1.97±0.11 Salt 1.54±0.13

Door 1.05±0.14 Center 1.60±0.18 Control 1.74±0.15 Fan 2.26±0.14 Side 1.79±0.13 PLT® 1.88±0.15

Litter, Week 5, West Middle 2.07±0.14 Waterer 1.98±0.13 Salt 1.76±0.15

Door 1.59±0.13 Center 0.98±0.17 Control 1.05±0.14 Fan 0.64±0.13 Side 1.03±0.12 PLT® 1.32±0.14

Litter, Week 7, East Middle 1.23±0.13 Waterer 1.45±0.12 Salt 1.08±0.14

Door 0.93±0.18 Center 1.41±0.22 Control 1.23±0.18 Fan 1.13±0.18 Side 0.91±0.16 PLT® 1.31±0.18

Litter, Week 7, West Middle 1.61±0.18 Waterer 1.35±0.16 Salt 1.13±0.18

Door 0.30±0.11 Center 0.72±0.14 Control 0.52±0.12 Fan 0.66±0.11 Side 0.25±0.10 PLT® 0.32±0.12

Soil, Week 7, East Middle 0.58±0.11 Waterer 0.56±0.10 Salt 0.70±0.12

Door 0.24±0.12 Center 0.43±0.15 Control 0.20±0.12 Fan 0.33±0.12 Side 0.12±0.11 PLT® 0.08±0.12

Soil, Week 7, West Middle 0.25±0.12 Waterer 0.28±0.11 Salt 0.53±0.12

87

Appendix G. Average total aerobic, coliform, and Clostridium perfringens counts and prevalence rate of Salmonella spp. in litter pre- and post- windrow composting Total Aerobic

Counts (log10 cfu/ml±SE)

Colifrom Counts (log10 cfu/ml±SE)

Clostridium perfringens Counts (log10 cfu/ml±SE)

Salmonella spp. incidence rate [no. positive/total (%)]b

House A Pre-windrow (n=10)

4.08±0.11 Undetectable 2.34±0.27 0/10 (0%)

Post-windrow (n=10)

6.37±0.14a Undetectable 1.56±0.09 0/10 (0%)

House B Pre-windrow (n=10)

6.08±0.04a Undetectable 2.36±0.01 0/10 (0%)

Post-windrow (n=10)

6.80±0.04 Undetectable 1.73±0.06 0/10 (0%)

House C Pre-windrow (n=10)

7.41±0.17 1.12±0.34 2.15±0.17 4/10 (40%)

Post-windrow (n=10)

6.58±0.18 Undetectable 1.95±0.27 1/10 (10%)

House D Pre-windrow (n=60)

6.89±0.09 22.2±7.62c 2.23±0.10 2/12 (17%)d

Post-windrow (n=12)

4.98±0.21 Undetectable 0.98±0.24 0/12 (0%)

All houses are affected by gangrenous dermatitis an=9, one sample was uncountable bSuspected Salmonella colonies were confirmed with API20E cUnit is cfu/ml because the counts were too low dn=12 instead of n=60, 1 sample represents 5 pooled original samples

88

Appendix H. Results of t-test between two means (pre- and post-windrow) for total aerobic, coliform. and Clostridium perfringens counts in litter samples Mean

Difference (log10

cfu/ml)

T-value P-value Interpretation

House A n=20

Total Aerobic Count -2.2890 -12.9 <0.0001 Significant Clostridium perfringens count 0.7864 2.72 0.0196 Significant House B n=20

Total Aerobic Count -0.7200 -12.7 <0.0001 Significant Clostridium perfringens count 0.6270 7.77 <0.0001 Significant House C n=20

Total Aerobic Count 0.8358 3.39 0.0032 Significant Clostridium perfringens count 0.3905 1.22 0.2394 Not significant House D n=72

Total Aerobic Count 1.9078 8.92 <0.0001 Significant Coliform Count 22.2a 2.91 0.0050 Significant Clostridium perfringens count 1.2439 5.17 <0.0001 Significant aUnit is cfu/ml because the counts were too low α = 0.05

89

Appendix I. Aerobic, coliform, enterbactericeae, and E. coli populations measured in poultry processing by various researchers Stages of Processing Total aerobic

population (log10 cfu/ml)

Coliform and Enterobacteriaceae population (log10 cfu/ml) C=coliform E=enterobacteriaceae

E. coli population (log10 cfu/ml)

Farm 1-day before processing: litter drag swab, 6.95d

1-day before processing: litter drag swab, (C) 4.24d

1-day before processing: litter drag swab, 3.88d

Transportation/initial level entering plant

Post-kill: carcass, 4.72a Post-kill: neck skin, 7.4 & 7.56c

Post-kill: carcass, 6.8g

Post-stun: carcass, 7.17 & 6.96h

Post-kill: breast skin, 6.52j

Post-kill: neck skin, 5.2m

Post-kill: neck skin, 6.4, 6, 5.2, 6.3 & 6.4p

Post-kill: neck skin, (C) 5.35 & 5.67c

Post-kill: neck skin, (E) 5.36 & 5.75c

Post-kill: carcass, (C) 5g

Post-stun: carcass, (C) 6.2 & 5.63h

Post-kill: breast skin, (E) 4.76j

Post-kill: neck skin, (E) 4.8m

Post-kill: neck skin, (C) 4.6m

Post-kill: neck skin, (C) 3.5, 3.4, 2.4, 3.8 & 3.4p

Post-kill: carcass, 4.6a

Post-kill: carcass, 4.3g

Post-stun: carcass, 5.93 & 5.36h

Post-kill: neck skin, 4.2m

Scalding Post-scald: carcass, 4.67a

Post-scald: neck skin, 6.87 & 7.28c

Post-scald: carcass, 5g

Post-scald: breast skin, 5.76j

Post-scald: carcass, 5.09 & 4.45l

Post-scald rinse: carcass, 4.57l

Post-scald: neck skin, 5.5, 5.7, 4.7, 5.6 & 5.9p

Post-scald: neck skin, (C) 4.76 & 5.01c

Post-scald: neck skin, (E) 5.04 & 4.76c

Post-scald: carcass, (C) 2.9g

Post-scald: breast skin, (E) 3.36j

Post-scald: carcass, (C) 2.56 & 1.8l

Post-scald rinse: carcass, (C) 1.91l

Post-scald: neck skin, (C) 2.8, 3.5, 2, 3.4 & 3.4p

Post-scald: carcass, 4.57a

Post-scald: carcass, 2.1g

Post-scald: carcass, 2.27 & 1.55l

Post-scald rinse: carcass, 1.65l

Defeathering Post-pick: neck Post-pick: neck skin, Post-pick:

90

skin, 5.98 & 5.87c

Post-pick: carcass, 5g

Post-pick: carcass, 4.1 & 3.95h

Post-pick: breast skin, 4.15j

Post-pick: neck skin, 7 & 7.2m

Pre-NYW: carcass, ~ 4.6 & 4.5o

Post-NYW: carcass, ~ 4.1 & 4.1o

Post-pick: neck skin, 5, 5.5, 4.6, 4.4 & 4.8p

(C) 4.53 & 4.27c

Post-pick: neck skin, (E) 4.34 & 4.09c

Post-pick: carcass, (C) 3.4g

Post-pick: carcass, (C) 3.25 & 2.99h

Post-pick: breast skin, (E) 2.54j

Post-pick: neck skin, (E) 5.5 & 5.6m Post-pick: neck skin, (C) 5.4 & 5.6m

Pre-NYW: carcass, (C) ~ 3.7 & 2.9o

Post-NYW: carcass, (C) ~ 3.2 & 2.8o

Post-pick: neck skin, (C) 3.3, 3.8, 2.2, 2.8 & 3p

carcass, 2.8g

Post-pick: carcass, 3.02 & 2.8h

Post-pick: neck skin, 4.9 & 4.8m

Pre-NYW: carcass, ~ 3.4 & 2.6o

Post-NYW: carcass, ~ 2.8 & 2.5o

Evisceration Pre-salvage: carcass, 4.63a

Post-salvage: carcass, 4.53a

Post-evisceration: neck skin, 5.76 & 5.69c

Pre-evisceration: carcass, 4.48d

Post-evisceration: carcass, 3.81d

Post-evisceration: carcass, 4.5g

Pre-evisceration: carcass, 3.98i

Post-evisceration: carcass, 4.11 & 4.39l

Post-evisceration: neck skin, 7.2 & 7.2m

Post-evisceration wash: carcass, ~ 4o

Post-evisceration: neck skin, 5, 5.2,

Post-evisceration: neck skin, (C) 4.6 & 4.42c

Post-evisceration: neck skin, (E) 4.55 & 3.90c

Pre-evisceration: carcass, (C) 3.91d

Post-evisceration: carcass, (C) 3.27d

Post-evisceration: carcass, (C) 3.1g

Pre-evisceration: carcass, (E) 3.22i

Post-evisceration: carcass, (C) 2.71 & 3.03l

Post-evisceration: neck skin, (E) 5.3 & 5.4m

Post-evisceration: neck skin, (C) 5.2 & 5.7m

Post-evisceration wash: carcass, (C) ~ 3.1o

Pre-salvage: carcass, 4.52a

Post-salvage: carcass, 4.44a

Pre-evisceration: carcass, 3.74d

Post-evisceration: carcass, 3.08d

Post-evisceration: carcass, 2.2g

Pre-evisceration: carcass, 2.72i

Post-evisceration: carcass, 2.47 & 2.82l

Post-evisceration: neck skin, 4.9 & 5m

Post-evisceration wash: carcass, ~ 2.7o

Pre-evisceration: carcass, 2.09q

91

4.9, 4.6 & 4.4p

Pre-evisceration: carcass, 3.73q

Post-evisceration: neck skin, (C) 3.5, 3.9, 2.7, 3.4 & 3.1p

Pre-evisceration: carcass, (E) 2.7q

Carcass washers Post-SW: neck skin, 5.1 & 4.95c

Post-IOBW: carcass, 3.6g

Pre-IOBW: carcass, 3.8, 5 & 4.4k

Post-IOBW: carcass, 3.4, 4.7 & 3.8k

Post-SW: neck skin, 6 & 6.3m

Post-IOBW: carcass, ~ 3.3 & 3.6o

Post-SW: neck skin, 4.6, 4.8, 4.4, 4.4 & 4.6p

Post-SW: neck skin, (C) 3.97 & 3.75c

Post-SW: neck skin, (E) 3.77 & 3.67c

Post-IOBW: carcass, (C) 2.2g

Pre-IOBW: carcass, (C) 2.6, 3.7 & 3.4k

Post-IOBW: carcass, (C) 2.4, 3.1 & 2.8k

Post-SW: neck skin, (E) 5.2 & 5.2m

Post-SW: neck skin, (C) 5.1 & 5.2m

Post-IOBW: carcass, (C) ~ 2.2 & 2.5o

Post-SW: neck skin, (C) 3.4, 3.6, 2.3, 3.1 & 3.1p

Pre-IOBW: carcass, 2.51 & 2.76b Post-IOBW: carcass, 1.45 & 2.80b

Post-IOBW: carcass, 1.5g

Pre-IOBW: carcass, 2.2, 3.3 & 3.1k

Post-IOBW: carcass, 2.1, 2.7 & 2.4k

Post-SW: neck skin, 1.9 & 2m

Post-IOBW: carcass, ~ 1.9 & 2o

Carcass reprocessing Pre-Cecure®: carcass, 4.6a

Post- Cecure®: carcass, 2.2a

Post-ASC: carcass, ~ 3.1o

Post-TSP: carcass, ~ 3.2 & 3.3o

Post-ASC: carcass, (C) ~ 1.8o

Post-TSP: carcass, (C) ~ 1.7 & 1.4o

Pre-Cecure®: carcass, 4.5a

Post- Cecure®: carcass, 1.96a

Post-ASC: carcass, ~ 1.4o

Post-TSP: carcass, ~ 1.4 & 1.1o

Carcass chilling Pre-chill: carcass, 1.97a

Post-chill: carcass, 1.95a

Post-chill: neck skin, 5.18 & 5.13c

Post-chill: carcass, 3.23d

Post-chill: carcass, 3.38f

Post-airchill: carcass, 3.31f

Post-chill:

Post-chill: neck skin, (C) 3.99 & 3.92c

Post-chill: neck skin, (E) 3.81 & 3.91c

Post-airchill: carcass, (C) 2.59d

Pre-chill: carcass, (C) 3.9e

Post-chill: carcass, (C) 2.6e

Post-chill: carcass, (C) 1.72f

Post-airchill: carcass,

Pre-chill: carcass, 1.84a

Post-chill: carcass, 1.83a Post-chill: carcass, 1.22 & 1.74b

Post-airchill: carcass, 2.2d

Pre-chill: carcass, 3.2e

Post-chill: carcass, 1.8e

92

carcass, 2.9g

Pre-chill: carcass, 3.2i

Post-chill: carcass, 2.51i

Pre-chill: breast skin, 3.91j

Post-chill: breast skin, 3.71j

Post-chill: carcass, 3.59 & 4.01l

Post-airchill: neck skin, 6 & 6.1m

Pre-chill: carcass, ~ 4.2n

Post-chill: carcass, ~ 3.25n

Post-Cl2 chill: carcass, ~ 2.7 & 2.9o

Post-ASC/Cl2 chill: carcass, ~ 2.1o

Post-chill: neck skin, 4.4, 5.1, 3.6, 4.4 & 4.7p

Pre-chill: carcass, 3.18q

Post-chill: carcass, 2.87q

(C) 1.97f

Post-chill: carcass, (C) 1.9g

Pre-chill: carcass, (E) 2.57i

Post-chill: carcass, (E) 1.75i

Pre-chill: breast skin, (E) 2.64j

Post-chill: breast skin, (E) 2.51j

Post-chill: carcass, (C) 2.25 & 2.6l

Post-airchill: neck skin, (E) 5.3 & 5m

Post-airchill: neck skin, (C) 4.3 & 5m

Post-Cl2 chill: carcass, (C) ~ 1.2 & 1.3o

Post-ASC/Cl2 chill: carcass, (C) ~ 0.8o

Post-chill: neck skin, (C) 2.8, 3.7, 3 & 3p

Pre-chill: carcass, (E) 2.25q

Post-chill: carcass, (E) 1.56q

Post-chill: carcass, 1.17f

Post-airchill: carcass, 1.43f

Post-chill: carcass, 1.1g

Pre-chill: carcass, 2.04i

Post-chill: carcass, 1.2i

Post-chill: carcass, 2.09 & 2.36l

Post-airchill: neck skin, 4.4 & 4.1m

Pre-chill: carcass, ~ 2.4n

Post-chill: carcass, ~ 1.2n

Post-Cl2 chill: carcass, ~ 0.8 & 1o

Post-ASC/Cl2 chill: carcass, ~ 0.6o

Pre-chill: carcass, 1.61q

Post-chill: carcass, 0.89q

Post chilling treatment

Post-chill ASC: carcass, 0b

Packaging/retail Post-pack: neck skin, 6.4 & 6.5m

Post-pack: neck skin, 4.5, 5.3, 4.4 & 5.1p

Post-pack: neck skin, (E) 5.4 & 5m

Post-pack: neck skin, (C) 5 & 4.8m

Post-pack: neck skin, (C) 2.7, 3.8, 3.3 & 3.4p

Post-pack: neck skin, 4 & 3.9m

aRussell (2005) bOyarzabal and others (2004) cGöksoy and others (2004) *The unit is log10 cfu/g of neck skin dFluckey and others (2003) eNorthcutt and others (2003a) fSanchez and others (2002)

93

gBerrang and Dickens (2000) hBuhr and others (2000) iJames and others (1992a) jLillard (1989) *The unit is log10 cfu/g of breast skin kNorthcutt and others (2003b) lWaldroup and others (1993b) mAbu-Ruwaida and others (1994) *The unit is log10 cfu/g of neck skin nBilgili and others (2002) oStopforth and others (2007) pMead and others (1993) *The unit is log10 cfu/g of neck skin qJames and others (1992b) General format: Stage: sample type, count Abbreviation: IOBW=Inside outside bird washer; SW=Spray washer; NYW=New York Wash (Chlorine Wash after defeathering); ASC=Acidified sodium chlorite; TSP=Trisodium phosphate *Carcass=carcass rinse; post-chill=post-immersion chlorine chiller; All washes are chlorine unless indicated

94

Appendix J. Salmonella spp. incidence and populations measured in poultry production and processing by various researchers Stages of Processing Incidence Populations (log10

MPN/ml) Farm 1-day before processing:

cecum, 22%c

Pre-8h feed withdrawal: crop, 0%, 0%, 0%, 0% & 10%g

Post-8h feed withdrawal: crop, 7.5%, 10%, 5%, 2.6% & 30%g

Pre-8h feed withdrawal: cecum, 0% & 2.5%g

Post-8h feed withdrawal: cecum, 7.5% & 17.5%g

1-day before processing: litter drag swab, 1.67c

Transportation/initial level entering plant

Post-kill: carcass, 92.3 %a

Post-kill: neck skin, 40% & 33.3%b

Post-stun: carcass, 59.4% & 43.8%f

Post-kill: whole carcass swab, 7%m

Scalding Post-scald: carcass, 90.4%a

Post-scald: neck skin, 33.3% & 33.3%b

Post-scald: carcass, 11.3% & 1.3%j

Post-scald rinse: carcass, 6.3%j

Defeathering Post-pick: neck skin, 60% & 40%b

Post-pick: carcass, 59.4% & 40.6%f

Post-pick: carcass, 23%h

Post-pick: whole carcass swab, 16%m

Post-pick: carcass: 1.2e

Evisceration Pre-salvage: carcass, 84.6%a

Post-salvage: carcass, 75%a

Post-evisceration: neck skin, 26.6% & 33.3%b

Pre-evisceration: carcass, 33%i

Pre-evisceration: carcass, ~1.35c

Post-evisceration: carcass, ~1.18c

95

Post-evisceration: carcass, 27.5% & 28.8%j

Pre-evisceration: whole carcass swab, 5%m

Post-evisceration: whole carcass swab, 11%m

Pre-evisceration: carcass, 24%p

Carcass washers Post-SW: neck skin, 26.6% & 20%b

Post-SW (H2O): whole carcass swab, 1%m

Pre-IOBW: carcass, 95%o

Pre-IOBW: carcass, 1.56o

Carcass reprocessing Pre-Cecure®: carcass, 84.6%a

Post- Cecure®: carcass, 17.3%a

Carcass chilling Pre-chill: carcass, 15.4%a

Post-chill: carcass, 13.5%a

Post-chill: neck skin, 33.3% & 26.6%b

Post-chill: carcass, 24.7%d

Post-airchill: carcass, 18.7%d

Pre-chill: carcass, 20%h

Post-chill: carcass, 19%h

Pre-chill: carcass, 43%i

Post-chill: carcass, 46%i

Post-chill: carcass, 27.5% & 30.4%j

Pre-chill: carcass enrichment, 88.4%k

Post-chill: carcass enrichment, 84.1%k

Pre-chill: carcass, 20.7%l

Post-chill: carcass, 5.7%l

Pre-chill: whole carcass swab, 10%m

Post-chill: whole carcass swab, 16%m

Pre-chill: carcass swab, 26% & 20%n

Post-chill: carcass swab, 6.4% & 13.7%n

Pre-chill: carcass, 100%o

Post-chill: carcass, 41.7%o

Post-airchill: carcass, ~0.94c

Pre-chill: carcass, 0.75o

Post-chill: carcass, -1.29o

96

Pre-chill: carcass, 28%p

Post-chill: carcass, 49%p

aRussell (2005) bGöksoy and others (2004) *The unit is log10 cfu/g of neck skin cFluckey and others (2003) dSanchez and others (2002) eCason and others (2000) fBuhr and others (2000) gCorrier and others (1999) hCason and others (1997) iJames and others (1992a) jWaldroup and others (1993b) kParveen and others (2007) lBilgili and others (2002) mNde and others (2006) nLogue and others (2003) oBrichta-Harhay and others (2007) pJames and others (1992b) General format: Stage: sample type, count Abbreviation: MPN=Most probable number; IOBW=Inside outside bird washer; SW=Spray washer; ASC=Acidified sodium chlorite *Carcass=carcass rinse; post-chill=post-immersion chlorine chiller; All washes are chlorine unless indicated

97

Appendix K. Average coliform, Salmonella spp. and Campylobacter spp. counts of carcass rinses taken from different stages of a poultry processing plant Coliform

Counts (log10 cfu/ml±SE) n=5

Salmonella spp. Counts (log10 cfu/ml±SE) n=5

Campylobacter spp. Counts (log10 cfu/ml±SE) n=5

Biochemical Identification of Presumptive Campylobactera

Receiving 5.47±0.20 0.56±0.27 5.29±0.08 I: 5 of 5 negative II: 5 of 5 negative III: 3 of 5 C. jejuni & 2 of 5 C. coli IV: 1 of 5 C. jejuni V: 1 of 5 C. lari

Post-scald 4.53±0.19 0.72±0.30 3.46±0.07 I: 5 of 5 C. lari II: 5 of 5 negative III: 5 of 5 negative IV: 1 of 5 C. jejuni V: 1 of 5 C. coli

Post-pick 3.64±0.30 0.30±0.28 2.75±0.26 I: 5 of 5 negative II: 5 of 5 negative III: 3 of 5 C. lari IV: 3 of 5 C. lari V: 5 of 5 negative

Rehang/pre-evisceration

2.95±0.16 undetectable 2.73±0.17 I: 5 of 5 negative II: 1 of 5 C. jejuni III: 5 of 5 negative IV: 5 of 5 negative V: 5 of 5 negative

Post-evisceration

3.71±0.25 undetectable 2.25±0.39 I: 5 of 5 negative II: 3 of 5 C. jejuni III: 5 of 5 negative IV: 5 of 5 negative V: 2 of 5 C. jejuni

Post-IOBW 3.07±0.23 undetectable 1.76±0.21 I: 2 of 5 C. jejuni & 1 of 5 C. coli II: 5 of 5 negative III: 5 of 5 negative IV: 1 of 5 C. jejuni V: 5 of 5 negative

Pre-SANOVA® 2.77±0.17 undetectable 1.53±0.31 I: 5 of 5 negative II: 5 of 5 negative III: 2 of 5 C. jejuni IV: 5 of 5 negative V: 2 of 5 C. jejuni

Post-SANOVA®

1.35±0.37 undetectable 0.41±0.17 I: 5 of 5 negative II: 5 of 5 negative

98

III: 5 of 5 negative IV: 5 of 5 negative V: 5 of 5 negative

Post-chill 0.38±0.28 undetectable 0.61±0.27 I: Not tested II: Not tested III: Not tested IV: 5 of 5 negative V: Not tested

Each coliform, Salmonella spp. and Campylobacter spp. count is an average of duplicates aFive presumptive colonies were taken from mCampy-Cefex media and tested biochemically for Campylobacter spp. Oxidase test serves as an initial screening test. All oxidase negative colonies are designated as negative (not C. jejuni, C. coli and C. lari). Oxidase positive colonies are tested against hippurate hydrolysis, naladixic acid resistance and cephalothin resistance.

99

Appendix L. Results of t-test between two means for coliform, Salmonella spp., and Campylobacter spp. counts of carcass rinse samples taken from different stages of a poultry processing line Mean

Difference (log10 cfu/ml) n=10

T-value P-value Interpretation

Coliform Counts Receiving – Post-scald 0.9484 3.39 0.0094 Significant Post-scald – Post-pick 0.8834 2.38 0.0443 Significant Rehang – Post-evisceration -0.7680 -2.49 0.0378 Significant Post-evisceration – Post-IOBW 0.6413 1.79 0.1110 Not significant Pre-SANOVATM - Post-SANOVATM

1.4189 3.87 0.0048 Significant

Post-SANOVATM - Post-chill 0.9737 2.31 0.0499 Significant Salmonella spp. Counts Receiving – Post-scald -0.1600 -0.41 0.6956 Not significant Post-scald – Post-pick 0.4250 1.01 0.3417 Not significant Campylobacter spp. Counts Receiving – Post-scald 1.8321 16.43 <0.0001 Significant Post-scald – Post-pick 0.7068 2.89 0.0201 Significant Rehang – Post-evisceration 0.4861 3.33 0.0104 Significant Post-evisceration – Post-IOBW 0.4866 2.19 0.0600 Not significant Pre-SANOVATM - Post-SANOVATM

1.1204 4.74 0.0015 Significant

Post-SANOVATM - Post-chill -02030 -0.64 0.5390 Not significant α = 0.05

100

Appendix M. Average total aerobic and coliform counts of carcass rinses taken from pre-OLR, post-OLR, and post-chill stages of a processing plant using two types of OLR antimicrobial solutions Total Aerobic Count

(log10 cfu/ml±SE) Coliform Count (log10 cfu/ml±)

Day Line Pre- OLR n=10

Post-OLR n=10

Post-chill n=10

Pre- OLR n=10

Post-OLR n=10

Post-chill n=10

1 1 4.12± 0.16

3.80± 0.19

1.67± 0.18a

3.40± 0.15

2.65± 0.18

0.50± 0.21a

2 1 3.85± 0.08

2.90± 0.10

1.92± 0.15

2.38± 0.10

0.52± 0.17

0.06± 0.06

3 1 4.09± 0.12

3.13± 0.12

1.55± 0.16

2.91± 0.17

0.36± 0.12

0.90± 0.06

4 1 4.15± 0.12

3.37± 0.18

1.88± 0.14

2.95± 0.18

1.09± 0.24

0.59± 0.18

Line 1 Total n=40

4.05± 0.06

3.30± 0.09

1.76± 0.08b

2.91± 0.09

1.15± 0.17

0.31± 0.08b

1 2 4.50± 0.15

3.92± 0.11

1.55± 0.23

3.93± 0.18

2.98± 0.16

0.09± 0.05

2 2 3.78± 0.09

3.93± 0.15

1.57± 0.15

2.40± 0.15

2.93± 0.24

0.00± 0.00

3 2 4.02± 0.05

3.67± 0.08

1.73± 0.08

2.72± 0.10

2.34± 0.12

0.07± 0.07

4 2 3.93± 0.10

3.59± 0.14

1.81± 0.10

2.79± 0.13

2.05± 0.26

0.40± 0.09

Line 2 Total n=40

4.06± 0.07

3.78± 0.06

1.66± 0.07

2.96± 0.12

2.56± 0.12

0.14± 0.04

SANOVATM Total n=80

4.06± 0.05

3.54± 0.06

1.71± 0.05c

2.93± 0.07

1.86± 0.13

0.22± 0.04c

5 1 3.75± 0.08

3.25± 0.15

1.64± 0.23

2.71± 0.11

0.56± 0.14

0.55± 0.22

6 1 4.61± 0.20

3.31± 0.08

1.72± 0.16

3.37± 0.21

0.92± 0.13

0.37± 0.12

7 1 4.07± 0.06

3.69± 0.09

1.71± 0.15

2.72± 0.06

1.68± 0.13

0.44± 0.12

8 1 4.32± 0.15a

3.05± 0.08

2.15± 0.08

3.19± 0.16

0.78± 0.16

0.74± 0.12

Line 1 Total n=40

4.18± 0.08b

3.33± 0.06

1.81± 0.09

3.00± 0.08

0.98± 0.10

0.52± 0.08

5 2 3.89± 0.10

3.91± 0.17

1.79± 0.17

2.94± 0.15

2.82± 0.18

0.37± 0.15

6 2 5.12± 0.23

4.45± 0.15

1.29± 0.23

4.32± 0.21

3.59± 0.19

0.46± 0.16

7 2 3.96± 0.10

4.17± 0.15

1.60± 0.13

2.97± 0.14

3.14± 0.20

0.34± 0.14

101

8 2 4.46± 0.13

3.93± 0.13

2.12± 0.23

3.28± 0.18

2.58± 0.20

0.09± 0.06

Line 2 Total n=40

4.36± 0.11

4.11± 0.08

1.70± 0.11

3.38± 0.12

3.03± 0.11

0.32± 0.07

PERASAFE® Total n=80

4.27± 0.07c

3.72± 0.07

1.75± 0.07

3.19± 0.08

2.01± 0.14

0.42± 0.05

an=9 instead of n=10 bn=39 instead of n=40 cn=79 instead of n=80

102

Appendix N. Comparison of total aerobic and coliform count reductions between two antimicrobial solutions Mean Difference

(log10 cfu/ml) T-value P-value Interpretation

Line 1 (TAC) n=160

(Pre-OLRS – Post-OLRS) – (Pre-OLRP – Post-OLRP)a

-0.05492867 -0.73 0.4650 Not Significant

(Pre-OLRS – Post-chillS) – (Pre-OLRP – Post-chillP)b

-0.04163848 -0.53 0.5956 Not Significant

(Post-OLRS – Post-chillS) – (Post-OLRP – Post-chillP)a

0.01329018 0.16 0.8693 Not Significant

Line 2 (TAC) n=160

(Pre-OLRS – Post-OLRS) – (Pre-OLRP – Post-OLRP)

0.02024509 0.25 0.8033 Not Significant

(Pre-OLRS – Post-chillS) – (Pre-OLRP – Post-chillP)

-0.13248937 -0.14 0.1431 Not Significant

(Post-OLRS – Post-chillS) – (Post-OLRP – Post-chillP)

-0.15273445 -1.86 0.0654 Not Significant

Both Lines (TAC) n=320

(Pre-OLRS – Post-OLRS) – (Pre-OLRP – Post-OLRP)c

-0.01674464 -0.29 0.7711 Not Significant

(Pre-OLRS – Post-chillS) – (Pre-OLRP – Post-chillP)d

-0.08735326 -1.46 0.1448 Not Significant

(Post-OLRS – Post-chillS) – (Post-OLRP – Post-chillP)c

-0.06858802 -1.12 0.2639 Not Significant

Line 1 (Coliform Count) n=160

(Pre-OLRS – Post-OLRS) – (Pre-OLRP – Post-OLRP)

-0.12774459 -1.10 0.2743 Not Significant

(Pre-OLRS – Post-chillS) – (Pre-OLRP – Post-chillP)a

0.06487419 0.78 0.4390 Not Significant

(Post-OLRS – Post-chillS) – (Post-OLRP – Post-chillP)a

0.19261878 1.72 0.0874 Not Significant

Line 2 (Coliform Count)

103

n=160 (Pre-OLRS – Post-OLRS) – (Pre-OLRP – Post-OLRP)

0.01870484 0.16 0.8722 Not Significant

(Pre-OLRS – Post-chillS) – (Pre-OLRP – Post-chillP)

-0.12004058 -1.30 0.1961 Not Significant

(Post-OLRS – Post-chillS) – (Post-OLRP – Post-chillP)

-0.13874541 -1.56 0.1219 Not Significant

Both Lines (Coliform Count) n=320

(Pre-OLRS – Post-OLRS) – (Pre-OLRP – Post-OLRP)

-0.06077277 -0.65 0.5162 Not Significant

(Pre-OLRS – Post-chillS) – (Pre-OLRP – Post-chillP)c

-0.02700972 -0.43 0.6705 Not Significant

(Post-OLRS – Post-chillS) – (Post-OLRP – Post-chillP)c

0.02992496 0.33 0.7407 Not Significant

an=159 instead of n=160 bn=158 instead of n=160 cn=319 instead of n=320 dn=318 instead of n=320 α = 0.05 Abbreviation: TAC=Total aerobic count; OLR=Online reprocessing; Subscript S=SANOVATM; Subscript P=PERASAFE®

104

Appendix O. GLM table for total aerobic and coliform counts in carcass rinses taken at pre-OLR stage of poultry processing Source Mean Square F-value P-value Interpretation Total Aerobic Count

Line 0.32401087 1.97 0.1625 Not significant Day 2.42488875 14.75 <0.0001 Significant Line*Day 0.32046233 1.95 0.0660 Not significant Coliform Count

Line 1.82951114 7.66 0.0064 Significant Day 4.76338507 19.95 <0.0001 Significant Line*Day 0.71360331 2.99 0.0059 Significant α=0.05

105

Appendix P. Least mean squares of total aerobic and coliform counts in carcass rinses taken at pre-OLR stage of poultry processing based on line and day of collection Line log10

cfu/ml±SE Day log10

cfu/ml±SE Line*Day log10

cfu/ml±SE 1 4.12±0.05 1 4.31±0.09 1*1 4.12±0.13 2 4.21±0.05 2 3.81±0.09 1*2 3.85±0.13

3 4.06±0.09 1*3 4.09±0.13 4 4.04±0.09 1*4 4.15±0.13 5 3.82±0.09 1*5 3.75±0.13 6 4.86±0.09 1*6 4.61±0.13 7 4.02±0.09 1*7 4.07±0.13 8 4.39±0.09 1*8 4.32±0.13

2*1 4.50±0.13 2*2 3.78±0.13 2*3 4.02±0.13 2*4 3.93±0.13 2*5 3.89±0.13 2*6 5.12±0.13 2*7 3.96±0.13

Total Aerobic Count

2*8 4.46±0.13 1 2.95±0.05 1 3.67±0.11 1*1 3.40±0.15 2 3.17±0.05 2 2.39±0.11 1*2 2.38±0.15

3 2.81±0.11 1*3 2.91±0.15 4 2.87±0.11 1*4 2.95±0.15 5 2.83±0.11 1*5 2.71±0.15 6 2.85±0.11 1*6 3.37±0.15 7 2.84±0.11 1*7 2.72±0.15 8 3.23±0.11 1*8 3.19±0.15

2*1 3.93±0.15 2*2 2.40±0.15 2*3 2.72±0.15 2*4 2.79±0.15 2*5 2.94±0.15 2*6 4.32±0.15 2*7 2.97±0.15

Coliform Count

2*8 3.28±0.15 Different flocks are slaughtered on different days.

106

Bibliography

Abu-Ruwaida, A. S., W. N. Sawaya, B. H. Dashti, M. Murad and H. A. Al-Othman. 1994. Microbiological quality of broilers during processing in a modern commercial slaughterhouse in Kuwait. Journal of Food Protection. 57:887-892.

Anonymous. No date. Perasafe direct meat intervention for meat & poultry industry

(online advertisement). Available at: http://www.afcocare.com. Accessed 26 October 2007.

Al Homidan, A. A., J. F. Robertson and A. M. Petchey. 2003. Review of the effect of

ammonia and dust concentrations on broiler performance. Worlds Poultry Science. 59:340-349.

Altekruse, S. F. and D. L. Swerdlow. 2002. Campylobacter jejuni and related

organisms. In Cliver, D. O. and H. P. Riemann (eds.). Foodborne Diseases, 2nd ed. Academic Press.: Barcelona, 103-112.

Atkinson, C. F., D. D. Jones and J. J. Gauthier. 1996. Biodegradability and microbial

activities during composti of poultry litter. Poultry Science. 75:608-617. Bashor, M. P., P. A. Curtis, K. M. Keener, B. W. Sheldon, S. Kathariou and J. A.

Osborne. 2004. Effects of carcass washers on Campylobacter contamination in large broiler processing plants. Poultry Science. 83:1232-1239.

Beker, A., S. L. Vanhooser, J. H. Swartzlander and R. G. Teeter. 2004. Atmospheric

ammonia concentration effects on broiler growth and performance. Poultry Science. 13:5-9.

Bennett, P. 2006. Compliance guideline for controlling Salmonella in poultry. 1st ed.

Food Safety and Inspection Services. Available at: http://www.fsis.usda.gov/PDF/Compliance_Guideline_Controlling_Salmonella_Poultry.pdf. Accessed 13 August 2007.

Berkelman, R. and M. Doyle. 2006. ASM submits comments on FSIS Salmonella

verification reporting. Available at: http://www.asm.org/Policy/index.asp?bid=43091 Accessed 11 February 2008.

Berrang, M. E., J. S. Bailey, S. F. Altekruse, B. Patel, W. K. Shaw, Jr., R. J.

Meinermann and P. J. Fedorka-Cray. 2007. Prevalence and numbers of

107

Campylobacter on broiler carcasses collected at rehang and postchill in 20 U.S. processing plants. Journal of Food Protection. 70:1556-1560.

Berrang, M. E. and J. A. Dickens. 2000. Presence and level of Campylobacter spp. on

broiler carcasses throughout the processing plant. Journal of Applied Poultry Research. 9:43-47.

Beutin, L., D. Geier, H. Steinrück, S. Zimmermann and F. Scheutz. 1993. Prevalence

and some properties of verotoxin (shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. Journal of Clinical Microbiology. 31:2483-2488.

Bilgili, S. F., A. L. Waldroup, D. Zelenka and J. E. Marion. 2002. Visible ingesta on

prechill carcasses does not affect the microbiological quality of broiler carcasses after immersion chilling. Journal of Applied Poultry Research. 11:233-238.

Blake, J. P. and J. B. Hess. 2001. Sodium bisulfate (PLT) as a litter treatment.

Alabama Cooperative Extension System. Available at: http://www.aces.edu/pubs/docs/A/ANR-1208/. Accessed 21 May 2007.

Brichta-Harhay, D. M., T. M. Arthur and M. Koohmaraie. 2007. Enumeration of

Salmonella from poultry carcass rinses via direct plating methods. Letters in Applied Microbiology. 46:186-191.

Brodie, H. L., L. E. Carr and P. Condon. No date. Poultry litter compost production, a

means of distributing excess nutrients. Available at: http://www.p2pays.org/ref/12/11556.pdf Accessed: 7 February 2008.

Buhr, R. J., J. A. Cason, J. A. Dickens, A. Hinton, Jr. and K. D. Ingram. 2000.

Influence of flooring type during transport and holding on bacteria recovery from broiler carcass rinses before and after defeathering. Poultry Science. 79:436-441.

Byrd, J. A., D. E. Corrier, M. E. Hume, R. H. Bailey, L. H. Stanker and B. M. Hargis.

1998. Effect feed withdrawal on Campylobacter in the crops of market-age broiler chickens. Avian Diseases. 42:802-806.

Cason, J. A., J. S. Bailey, N. J. Stern, A. D. Whittemore and N. A. Cox. 1997.

Relationship between aerobic bacteria, Salmonellae and Campylobacter on broiler carcass. Poultry Science. 76:1037-1041.

Cason, J. A., A. Hinton, Jr. and K. D. Ingram. 2000. Coliform, Escherichia coli, and

Salmonellae concentrations in a multiple-tank, counterflow poultry scalder. Journal of Food Protection. 63:1184-1188.

108

Cason, J. A., A. D. Whittemore and A. D. Shackleford. 1999. Aerobic bacteria and solids in a three-tank, two-pass, counterflow scalder. Poutry Science. 78:144-147.

Caveny, D. D., C. L. Quarler and G. A. Greathouse. 1981. Atmospheric ammonia and

broiler cockerel performance. Poultry Science. 60:513-516. CDC. 2007. Salmonella Surveillance: Annual Summary, 2005. US Department of

Health and Human Services, Center for Disease Control. Available at: http://www.cdc.gov/Ncidod/dbmd/phlisdata/salmtab/2005/SalmonellaIntroduction2005.pdf Accessed: 1 November 2007.

Cervantes, H. M., L. L. Munger, D. H. Ley and M. D. Ficken. 1988. Staphylococcus-

induced gangrenous dermatitis in broilers. Avian Diseases. 32:140-142. Chantarapanont, W., M. E. Berrang and J. F. Frank. 2004. Direct microscopic

observation of viability of Campylobacter jejuni on chicken skin treated with selected chemical sanitizing agents. Journal of Food Protection. 67:1146-1152.

Clark, D., S. Watkins, F. Jones and B. Norton. 2004. Understanding and control of

gangrenous dermatitis in poultry houses. University of Arkansas Division of Agriculture Cooperative Extension Services. Available at: http://www.uaex.edu/Other_Areas/publications/PDF/FSA-7048.pdf Accessed: 9 November 2007.

Corrier, D. E., J. A. Byrd, B. M. Hargis, M. E. Hume, R. H. Bailey and L. H. Stanker.

1999. Presence of Salmonella in the crop and ceca of broiler chickens before and after preslaughter feed withdrawal. Poultry Science. 78:45-49.

Craven, S. E. 2001. Occurrence of Clostridium perfringens in broiler chicken

processing plant as determined by recovery in iron milk medium. Journal of Food Protection. 64:1956-1960.

Craven, S. E., N. A. Cox, J. S. Bailey and D. E. Cosby. 2003. Incidence and tracking

of Clostridium perfringens through an integrated broiler chicken operation. Avian Diseases. 47:707-711.

Craven, S. E., N. J. Stern, J. S. Bailey and N. A. Cox. 2001. Incidence of Clostridium

perfringens in broiler chickens and their environment during production and processing. Avian Diseases. 45:887-896.

D’Aoust, J.-Y., J. Maurer and J. S. Bailey. 2001. Salmonella species. In Doyle, M. P.,

L. R. Beuchat and T. J. Montville (eds.). Food Microbiology, 2nd ed. ASM Press: Washington, D.C., 141-178.

109

Dickens, J. A. and A. D. Whittemore. 1997. Effects of acetic acid and hydrogen peroxide application during defeathering on the microbiological quality of broiler carcasses prior to evisceration. Poultry Science. 76:657-660.

Dohms, J. E. 2003. Botulism. In Saif, Y. M., H. John Barnes, J. R. Glisson, A. M.

Fadly, L. R. McDougald and D. E. Swayne (eds.). Diseases of Poultry, 11th ed. Blackwell Publishing, Ltd.: Vermont, 785-791.

Feng, P., S. D. Weagant and M. A. Grant. 1998. Enumeration of Escherichia coli and

the coliform bacteria. In Bacteriological Analytical Manual Online. Available at: http://www.cfsan.fda.gov/~ebam/bam-4.html#authors Accessed: 1 November 2007.

Fletcher, D. L., E. W. Craig and J. W. Arnold. 1997. An evaluation of on-line

“reprocessing” on visual contamination and microbiological quality of broilers. Journal of Applied Poultry Research. 6:436-442.

Flory, G. A., E. S. Bendfeldt, R. W. Peer, C. Zirkle and G. W. Malone. Guidelines for

in-house composting poultry mortality as a rapid response to avian influenza. Virginia Cooperative Extension. Available at: http://www.deq.virginia.gov/waste/pdf/factsheet1va.pdf Accessed: 29 February 2008.

Fluckey, W. M., M. X. Sanchez, S. R. McKee, D. Smith, E. Pendleton and M. M.

Brashears. Establishment of a microbiological profile for an air-chilling poultry operation in the United States. Journal of Food Protection. 66:272-279.

Fratamico, P. M., J. L. Smith and R. L. Buchanan. 2002. Escherichia coli. In Cliver,

D. O. and H. P. Riemann (eds.). Foodborne Diseases, 2nd ed. Academic Press.: Barcelona, 79-101.

FSIS. 2007a. FSIS Nationwide Young Chicken Microbiological Baseline Data

Collection Program. Available at: http://www.fsis.usda.gov/PDF/Young_Chicken_Baseline_Data_Collection.pdf Accessed: 18 February 2008.

FSIS. 2007b. Serotype profile of Salmonella isolates from meat and poultry products:

January 1998 to December 2006. US Department of Agriculture, Food Safety and Inspection Services. Available at: http://www.fsis.usda.gov/PDF/Serotypes_Profile_Salmonella_Tables_&_Figures.pdf Accessed: 2 November 2007.

FSIS. 2005. Generic E. coli and Salmonella baseline results. Federal Register.

70:8058-8060. Available at:

110

http://www.fsis.usda.gov/OPPDE/rdad/FRPubs/02-046N.pdf Accessed: 11 February 2008.

FSIS, 1996a. Nationwide broiler chicken microbiological baseline data collection

program July 1994 – June 1995. Available at: http://www.fsis.usda.gov/OPHS/baseline/broiler1.pdf Accessed: 18 February 2008.

FSIS. 1996b. Pathogen reduction; hazard analysis and critical control point (HACCP)

systems; final rule. Federal Register. 61:38806-38989. Available at: http://www.fsis.usda.gov/OPPDE/rdad/FRPubs/93-016F.pdf Accessed: 8 February 2008.

Gast, R. K. 2003a. Introduction: salmonella infection. In Saif, Y. M., H. John Barnes,

J. R. Glisson, A. M. Fadly, L. R. McDougald and D. E. Swayne (eds.). Diseases of Poultry, 11th ed. Blackwell Publishing, Ltd.: Vermont, 567-568.

Gast, R. K. 2003b. Paratyphoid infection. In Saif, Y. M., H. John Barnes, J. R.

Glisson, A. M. Fadly, L. R. McDougald and D. E. Swayne (eds.). Diseases of Poultry, 11th ed. Blackwell Publishing, Ltd.: Vermont, 583-613.

Göksoy, E. Ö., Ş. Kirkan and F. Kök. 2004. Microbiological quality of broiler

carcasses during processing in two slaughterhouses in Turkey. Poultry Science. 83:1427-1432.

Gray, J. T. and P. J. Fedorka-Cray. 2002. Salmonella. In Cliver, D. O. and H. P.

Riemann (eds.). Foodborne Diseases, 2nd ed. Academic Press.: Barcelona, 55-68.

Haque, A. K. M. A. and J. M. Vandepopuliere. 1994. Composting cage layer manure

with poultry litter. Journal of Applied Poultry Research. 3:268-273. Hartel, P. G., W. I. Segars, J. D. Summer, J. V. Collins, A. T. Phillips and E. Whittle.

2000. Survival of fecal coliforms in fresh and stacked broiler litter. Journal of Applied Poultry Research. 9:505-512.

Heath, G. E., A. M. Thaler and W. O. James. 1994. A survey of stunning methods

currently used during slaughter of poultry in commercial poultry plants. Journal of Applied Poultry Research. 3:297-302.

Hinton, Jr., A., R. J. Buhr and K. D. Ingram. 2000. Reduction of Salmonella in the

crop of broiler chickens subjected to feed withdrawal. Poultry Science. 79:1566-1570.

Hunt, J. M., C. Abeyta and T. Tran. 1998. Campylobacter. In Bacteriological

Analytical Manual Online. Available at:

111

http://www.cfsan.fda.gov/~ebam/bam-4.html#authors Accessed: 3 March 2008.

Izat, A. L., F. A. Gardner, J. H. Denton and F. A. Golan. 1988. Incidence and level of

Campylobacter jejuni in broiler processing. Poultry Science. 67:1568-1572. James, W. O., R. L. Brewer, J. C. Prucha W. O. Williams and D. R. Parham. 1992a.

Effects of chlorination of chill water on the bacteriologic profile of raw chicken carcasses and giblets. Journal of American Veterinary Medical Association. 200:60-63.

James, W. O., J. C. Prucha, R. L. Brewer, W. O. Williams, Jr., W. A. Christensen, A.

M. Thaler and A. T. Hogue. 1992b. Effects of coutercurrent scalding and postscald spray on the bacteriologic profile of raw chicken carcasses. Journal of American Veterinary Medical Association. 201:705-708.

Jeffrey, J. S., J. H. Kirk, E. R. Atwill and J. S. Cullor. 1998. Prevalence of selected

microbial pathogens in processed poultry waste used as dairy cattle feed. Poultry Science. 77:808-811.

Jimenez, S. M., M. C. Tiburzi, M. S. Salsi, M. E. Pirovani and M. A. Moguuilevsky.

2003. The role of visible faecal material as a vehicle for generic Escherichia coli, coliform and other enterobacteria contaminating poultry carcasses during slaughtering. Journal of Applied Microbiology. 95:451-456.

Kahn, M (ed). 2005. The Merck Veterinary Manual. Merck & Co.,Inc.: New Jersey. Kariuki, S., C. Gilks, J. Kimari, J. Muyodi, B. Getty and C. A. Hart. 2002. Carriage of

potentially pathogenic Escherichia coli in chickens. Avian Diseases. 46:721-724.

Keener, K. M., M. P. Bashor, P. A. Curtis, B. W. Sheldon and S. Kathariou. 2004.

Comprehensive review of Campylobacter and poultry processing. Comprehensive Reviews in Food Science and Food Safety. 3:105-115.

Kelleher, B. P., J. J. Leahy, A. M. Henihan, T. F. O’Dwyer, D. Sutton and M. J.

Leahy. 2002. Advances in poultry litter disposal technology – a review. Bioresource Technology. 83:27-36.

Kemp, G. K., M. L. Aldrich, M. L. Guerra and K. R. Schneider. 2001. Continuous

online processing of fecal- and ingesta- contaminated poultry carcasses using an acidified sodium chlorite antimicrobial intervention. Journal of Food Protection. 64:807-812.

112

Kemp, G.K., M. L. Aldrich and A. L. Waldroup. 2000. Acidified sodium chlorite antimicrobial treatment of broiler carcasses. Journal of Food Protection. 63:1087-1092.

Kemp, G. K. and K. R. Schneider. 2000. Validation of thiosulfate for neutralization of

acidified sodium chlorite in microbiological testing. Poultry Science. 79:1857-1860.

Kling, H. F. and C. L. Quarles. 1974. Effect of atmospheric ammonia and the stress of

infectious bronchitis vaccination on Leghorn males. Poultry Science. 53:1161-1165.

Kobayashi, H., T. Pohjanvirta and S. Pelkonen. 2002. Prevalence and characteristics

of intimin- and shiga toxin-producing Escherichia coli from gulls, pigeons and broilers in Finland. Journal of Veterinary Medical Science. 64:1071-1073.

Labbe, R. G. and V. K. Juneja. 2002. Clostridium perfringens. In Cliver, D. O. and H.

P. Riemann. (eds.). Foodborne Diseases, 2nd ed. Academic Press.: Barcelona, 119-126.

Lavergne, T. K., M. F. Stephens, D. Schellinger and W. Carney, Jr.. 2006. In-house

pasteurization of broiler litter. Louisiana Cooperative Extension Services. Available at: http://www.lsuagcenter.com/NR/rdonlyres/696449C7-CA3F-4A92-9ED4-0C67587DCC6A/29454/pub2955InhousePastuerization.pdf Accessed: 28 February 2008.

Lillard, H. S. 1989. Incidence and recovery of Salmonellae and other bacteria from

commercially processed poultry carcasses at selected pre- and post- evisceration steps. Journal of Food Protection. 52:88-91.

Line, J. E. 2002. Campylobacter and Salmonella populations associated with chickens

raised on acidified litter. Poultry Science. 81: 1473-1477. Line, J. E. 2001. Development of a selective differential agar for isolation and

enumeration of Campylobacter spp. Journal of Food Protection. 64:1711-1715.

Line, J. E. and J. S. Bailey. 2006. Effect of on-farm litter acidification treatments on

Campylobacter and Salmonella populations in commercial broiler houses in northeast Georgia. Poultry Science. 85:1529-1534.

Line, J. E., J. S. Bailey, N. A. Cox and N. J. Stern. 1997. Yeast treatment to reduce

Salmonella and Campylobacter populations associated with broiler chickens subjected to transport stress. Poultry Science. 76:1227-1231.

113

Line, J. E., N. J. Stern, C. P. Lattuada and S. T. Benson. 2001. Comparison of methods for recovery and enumeration of Campylobacter from freshly processed broilers. Journal of Food Protection. 64:982-986.

Logue, C. M., J. S. Sherwood, P. A. Olah, L. M. Elijah and M. R. Dockter. 2003. The

incidence of antimicrobial-resistant Salmonella spp. on freshly processed poultry from US Midwestern processing plants. Journal of Applied Microbiology. 94:16-24.

McClane, B. A. 2001. Clostridium perfringens. In Doyle, M. P., L. R. Beuchat and T.

J. Montville (eds.). Food Microbiology, 2nd ed. ASM Press: Washington, D.C., 351-372.

Macklin, K. 2007. In-house windrow composting presentation. Delmarva Breeder

Hatchery and Grow-out Conference. Available at: http://www.rec.udel.edu/Poultry/proceedings2007/Macklin_In-House%20Windrowing.pdf Accessed: 28 February 2008.

Macklin, K. S., J. B. Hess and S. F. Bilgili. 2008. In-house windrow composting and

its effects on foodborne pathogens. Journal of Applied Poultry Research. 17:121-127.

Macklin, K., G. Simpson, J. Donald and J. Campbell. 2007. Windrow composting of

litter to control disease-causing pathogens. The Poultry Engineering, Economics & Management Newsletter. Available at: http://www.aces.edu/dept/poultryventilation/documents/Nwsltr-47WindrowComposting.pdf Accessed: 28 February 2008.

Macklin, K. S., J. B. Hess, S. F. Bilgili and R. A. Norton. 2006. Effects of in-house

composting of litter on bacterial levels. Journal of Applied Poultry Research. 15:531-537.

Martin, S. A., M. A. McCann and W. D. Waltman II. 1998. Microbiological survey of

Georgia poultry litter. Journal of Applied Poultry Research. 7:90-98. May, K. N. 1974. Changes in microbial numbers during final washing and chilling of

commercially slaughtered broilers. Poultry Science. 53: 1282-1285. Mead, G. C., W. R. Hudson and M. H. Hinton. 1993. Microbiological survey of five

poultry processing plants in the UK. British Poultry Science. 34:497-503. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M.

Griffin and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerging Infectious Disease. 5:607-625. Available at: http://www.cdc.gov/ncidod/eid/vol5no5/pdf/mead.pdf Accessed: 29 October 2007.

114

Meng, J., M. P. Doyle, T. Zhao and S. Zhao. 2001. Enterohemorrhagic Escherichia

coli. In Doyle, M. P., L. R. Beuchat and T. J. Montville (eds.). Food Microbiology, 2nd ed. ASM Press: Washington, D.C., 193-213.

Moum, S. G., W. Seltzer and T. M. Goldhaft. 1969. A simple method of determining

concentrations of ammonia in animal quarters. Poultry Science. 48:347-348. Nachamkin, I. 2001. Campylobacter jejuni. In Doyle, M. P., L. R. Beuchat and T. J.

Montville (eds.). Food Microbiology, 2nd ed. ASM Press: Washington, D.C., 179-192.

Nagaraj, M., C. A. P. Wilson, B. Saenmahayak, J. B. Hess and S. F. Bilgili. 2007.

Efficacy of a litter amendment to reduce pododermatitis in broiler chickens. Journal of Applied Poultry Research. 16:255-261.

Nde, C. W., J. S. Sherwood, C. Doetkott and C. M. Logue. 2006. Prevalence and

molecular profiles of Salmonella collected at a commercial turkey processing plant. Journal of Food Protection. 69:1794-1801.

Neumann, T., D. Ritter, S. Dunham, J. Skalecki and Rehberger. 2006.

Characterization of Clostridium from broiler farms experiencing recurrent outbreaks of gangrenous dermatitis. Poultry Science Association Meeting. Available at: http://www.agtechproducts.com/research_poultry.htm Accessed on: 11 March 2008.

Northcutt, J. K., M. E. Berrang, J. A. Dickens, D. L. Fletcher and N. A. Cox. 2003a.

Effect of broiler age, feed withdrawal, and transportation on levels of coliforms, Campylobacter, Escherichia coli and Salmonella on carcasses before and after immersion chilling. Poultry Science. 82:169-173.

Northcutt, J. K., M. E. Berrang, D. P. Smith and D. R. Jones. 2003b. Effect of

commercial bird washers on broiler carcass microbiological characteristics. Journal of Applied Poultry Research. 12:435-438.

Omeira, N., E. K. Barbour, P. A. Nehme, S. K. Hamadeh, R. Zurayk and I. Bashour.

2006. Microbiological and chemical properties of liter from different chicken types and production systems. Science of the Total Environment. 367: 156-162.

Oyarzabal, O. A., K. S. Macklin, J. M. Barbaree and R. S. Miller. 2005. Evaluation of

agar plates for direct enumeration of Campylobacter spp. from poultry carcass rinses. Applied and Environmental Microbiology. 71:3351-3354.

Oyarzabal, O. A., C. Hawk, S. F. Bilgili, C. C. Warf and K. Kemp. 2004. Effects of

postchill application of acidified sodium chlorite to control Campylobacter

115

spp. and Escherichia coli on commercial broiler carcasses. Journal of Food Proctection. 67:2288-2291.

Oyarzabal, O. A. 2005. Reduction of Campylobacter spp. by commercial

antimicrobials applied during the processing of broiler chickens: a review from the United States perspective. Journal of Food Protection. 68:1752-1760.

Parveen, S., M. Taabodi, J. g. Schwarz, T. P. Oscar, J. Harter-Dennis and D. G.

White. 2007. Prevalence and antimicrobial resistance of Salmonella recovered from processed poultry. Journal of Food Protection. 70:2466-2472.

Payne, J. B., E. C. Kroger and S. E. Watkins. 2002. Evaluation of litter treatments on

Salmonella recovery from poultry litter. Journal of Applied Poultry Research. 11:239-243.

Pope, M. J. and T. E. Cherry. 2000. An evaluation of the presence of pathogens on

broilers raised on Poultry Litter Treatment®-treated litter. Poultry Science. 79:1351-1355.

Quarles, C. L. and D. D. Caveny. 1979. Effect of air contaminants on performance

and quality of broilers. Poultry Science. 58:543-548. Ramirez, G. A., L.L. Sarlin, D. J. Caldwell, C. R. Yezak, Jr., M. E. Hume, D. E.

Corrier, J. R. Deloach and B. M. Hargis. 1997. Effect of feed withdrawal on the incidence of Salmonella in crops and ceca of market age broiler chickens. Poultry Science. 76:654-656.

Rasekh, J., A. M. Thaler, D. L. Engeljohn and N. H. Pihkala. 2005. Food Safety and

Inspection Service policy for control of poultry contaminated by digestive tract contents: a review. Journal of Applied Poultry Research. 14:603-611.

Ritz, C. W., B. D. Fairchild and M. P. Lacy. 2004. Implications of ammonia

production and emissions from commercial poultry facilities: a review. Journal of Applied Poultry Research. 13:684-692.

Russell, S. M. 2007a. A Bar Too High?. Watt Poultry USA. 32-34. Russell, S. M. 2007b. Managing antimicrobials and application system. Watt Poultry

USA. 18-22. Russell, S. M. 2007c. Using Post-chill dips and sprays. Watt Poultry USA. 26. Russell, S. 2005. Verification of intervention strategies. Watt Poultry USA. 40-61. Russell, S. M. 2002. Intervention strategies for reducing Salmonella prevalence on

ready-to-cook chicken. University of Georgia College of Agriculture and

116

Environmental Sciences Cooperative extension Service. Available at: http://www.pubs.caes.uga.edu/caespubs/pubcd/b1222.htm. Accessed 9 August 2007.

Russell, S. M. 2000. Comparison of the traditional three-tube most probable number

method with the Petrifilm, SimPlate, BioSys Optical and Bactometer conductance methods for enumerating Escherichia coli from chicken carcasses and ground beef. Journal of Food Protection. 63:1179-1183.

Russell, S. and K. Keener. 2007. Chlorine: the misunderstood pathogen reduction

tool. Watt Poultry USA. 22-27. Russell, S. M. and J. M. Walker. 1997. The effect of evisceration on visible

contamination and the microbiological profile of fresh broiler chicken carcasses using the Nu-Tech evisceration system or the conventional streamlined inspection system. Poultry science. 76:780-784.

Sanchez, M. X., W. M. Fluckey, M. M. Brashears and S. R. McKee. 2002. Microbial

profile and antibiotic susceptibility of Campylobacter spp. and Salmonella spp. in broilers processed in air-chilled and immersion-chilled environments. Journal of Food Protection. 65:948-956.

Seo, K. H., I. E. Valentín-Bon and R. E. Brackett. 2006. Detection and enumeration

of Salmonella enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods. Journal of Food Protection. 69:639-643.

Shah, S., P. Westerman and J. Parsons. 2006. Poultry litter ammendments. North

Carolina Cooperative Extension Services. Available at: http://www.bae.ncsu.edu/programs/extension/publicat/wqwm/poultry/factsheet_agw-657long.pdf. Accessed 21 May 2007.

Shahidi, S. A. and A. R. Ferguson. 1971. New quantitative, qualitative and

confirmatory media for rapid analysis of food for Clostridium perfringns. Applied Microbiology. 21:500-506.

Simmons, M., D. L. Fletcher, J. A. Cason and M. E. Berrang. 2003. Recovery of

Salmonella from retail broilers by a whole carcass enrichment procedure. Journal of Food Protection. 66:446-450.

Smith, D. P., J. K. Northcutt, J. A. Cason, A. Hinton Jr., R. J. Buhr and K. D. Ingram.

2007. Effect of external or internal fecal contamination on numbers of bacteria on prechilled broiler carcasses. Poultry Science. 86:1241-1244.

117

Smith, D. P., J. K. Northcutt and M. T. Musgrove. 2005. Microbiology of contaminated or visibly clean broiler carcasses processed with an inside-outside bird washer. International Journal of Poultry Science. 4:955-958.

Stopforth, J. D., R. O’Connor, M. Lopes, B. Kottapalli, W. E. Hill and M.

Samadpour. 2007. Validation of individual and multiple-sequential interventions for reduction of microbial populates during processing of poultry carcasses and parts. Journal of Food Protection. 70:1393-1401.

Tablante, N. L., L. E. Carr, G. W. Malone, P. H. Patterson, F. N. Hegngi, G. Felton

and N. Zimmermann. 2002. Guidelines for in-house composting of catastrophic poultry mortality. Maryland Cooperative Extension. Available at: http://extension.umd.edu/publications/PDFs/FS801.pdf Accessed: 29 February 2008.

Terzich, M., C. Quarles, M. A. Goodwin and J. Brown. 1998a. Effect of Poultry Litter

Treatment® (PLT®) on death due to ascites in broilers. Avian Diseases. 42:385-387.

Terzich, M., C. Quarles, M. A. Goodwin and J. Brown. 1998b. Effect of Poultry

Litter Treatment® (PLT®) on the development of respiratory tract lesions in broilers. Avian Pathology. 27:566-569.

Terzich, M., M. J. Pope, T. E. Cherry and J. Hollinger. 2000. Survey of pathogens in

poultry litter in the United States. Journal of Applied Poultry Research. 9:287-291.

Valentín-Bon, I. E., R. E. Brackett, K. H. Seo, T. S. Hammack and W. H. Andrews.

2003. Preenrichment versus direct selective agar plating for the detection of Salmonella enteritidis in shell eggs. Journal of Food Protection. 66:1670-1674.

Vizzier Thaxton, Y., C. L. Balzli and J. D. Tankson. 2003. Relationship of broiler

flock number to litter microflora. Journal of Applied Poultry Research. 12:81-84.

Waldroup, A., B. Rathgeber and N. Imel. 1993. Microbiological aspects of counter

current scalding. Journal of Applied Poultry Research. 2:203-207. Wabeck, C. J. 2002a. Microbiology of poultry meat products. In Bell, D. D. and W.

D. Weaver, Jr. (eds.). Commercial Chicken Meat and Egg Production, 5th ed. Kluwer Academic Publishers.: Massachusetts, 889-898.

Wabeck, C. J. 2002b. Processing chicken meat. In Bell, D. D. and W. D. Weaver, Jr.

(eds.). Commercial Chicken Meat and Egg Production, 5th ed. Kluwer Academic Publishers.: Massachusetts, 899-920.

118

Wabeck, C. J. 2002c. Quality assurance and food safety---chicken meat. In Bell, D.

D. and W. D. Weaver, Jr. (eds.). Commercial Chicken Meat and Egg Production, 5th ed. Kluwer Academic Publishers.: Massachusetts, 871-887.

Wages, D. P. 2003. Ulcerative enteritis (quail disease). In Saif, Y. M., H. John

Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald and D. E. Swayne (eds.). Diseases of Poultry, 11th ed. Blackwell Publishing, Ltd.: Vermont, 775-781.

Wages, D. P. and K. Opengart. 2003a. Gangrenous dermatitis. In Saif, Y. M., H. John

Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald and D. E. Swayne (eds.). Diseases of Poultry, 11th ed. Blackwell Publishing, Ltd.: Vermont, 791-795.

Wages, D. P. and K. Opengart. 2003b. Necrotic enteritis. In Saif, Y. M., H. John

Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald and D. E. Swayne (eds.). Diseases of Poultry, 11th ed. Blackwell Publishing, Ltd.: Vermont, 781-785.

Waldroup, A. L., B. M. Rathgeber, R. E. Hierholzer, L. Smoot, L. M. Martin, S. F.

Bilgili, D. L. Fletcher, T. C. Chen and C. J. Wabeck. 1993a. Effects on reprocessing on microbiological quality of commercial prechill broiler carcasses. Journal of Applied Poultry Research. 2:111-116.

Waldroup, A. L., B. M. Rathgeber and N. Imel. 1993b. Microbiological aspect of

counter current scalding. Journal of Applied Poultry Research. 2:203-207. Wilder, T. D., J. M. Barbaree, K. S. Macklin and R. A. Norton. 2001. Differences in

the pathogenicity of various bacterial isolates used in an induction model for gangrenous dermatitis in broiler chickens. Avian Disease. 45:659-662.

Wilkinson, K. G. 2007. The biosecurity of on-farm mortality composting. Journal of

Applied Microbiology. 102: 609-618. Willoughby, D. H., A. A. Bickford, G. L. Cooper and B. R. Charlton. 1996. Periodic

recurrence of gangrenous dermatitis associated with Clostridium septicum in broiler chicken operation. Journal of Veterinary Diagnostic Investigation. 8:259-261.

Zhao, C., B. Ge, J. De Villena, R. Sudler, E. Yeh, S. Zhao, D. G. White, D. Wagner

and J. Meng. 2001. Prevalence of Campylobacter spp., Escherichia coli, and Salmonella serovars in retail chicken, turkey, pork, and beef from the greater Washington, D.C., area. Applied and Environmental Microbiology. 67: 5431-5436.

119

Yang, Z., Y. Li and M. Slavik. 1998. Use of antimicrobial spray applied with an inside-outside birdwasher to reduce bacterial contamination on prechilled chicken carcasses. Journal of Food Protection. 61:829-832.